U.S. patent number 7,842,729 [Application Number 11/145,499] was granted by the patent office on 2010-11-30 for anti tubercular drug: compositions and methods.
This patent grant is currently assigned to N/A, Sequella, Inc., The United States of America as represented by the Department of Health and Human Services. Invention is credited to Clifton E. Barry, III, Elena Bogatcheva, Leo Einck, Richard Edward Lee, Marina Nikolaevna Protopopova, Richard Allan Slayden.
United States Patent |
7,842,729 |
Protopopova , et
al. |
November 30, 2010 |
Anti tubercular drug: compositions and methods
Abstract
Methods and compositions for treating disease caused by
infectious agents, particularly tuberculosis. In particular,
methods and compositions comprising substituted ethylene diamines
for the treatment of infectious diseases are provided. In one
embodiment, these methods and compositions are used for the
treatment of mycobacterial infections, including, but not limited
to, tuberculosis.
Inventors: |
Protopopova; Marina Nikolaevna
(Silver Springs, MD), Lee; Richard Edward (Cordova, TN),
Slayden; Richard Allan (Ft. Collins, CO), Barry, III;
Clifton E. (Washington, DC), Bogatcheva; Elena
(Bethesda, MD), Einck; Leo (McLean, VA) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (Rockville, MD)
Sequella, Inc. (N/A)
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Family
ID: |
29552723 |
Appl.
No.: |
11/145,499 |
Filed: |
June 3, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060020041 A1 |
Jan 26, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10441146 |
May 19, 2003 |
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10147587 |
May 17, 2002 |
6951961 |
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60381220 |
May 17, 2002 |
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Current U.S.
Class: |
514/648; 514/306;
514/659; 514/654; 514/317; 564/316; 564/368; 564/355; 514/653;
546/134; 564/457; 564/454; 564/370; 564/369; 514/649; 564/320;
564/455; 514/660; 514/655; 564/366; 546/194; 546/246; 564/453 |
Current CPC
Class: |
A61P
31/10 (20180101); A61P 31/00 (20180101); A61P
31/04 (20180101); A61P 33/00 (20180101); A61P
31/06 (20180101); C07C 215/30 (20130101); A61K
31/137 (20130101); C07C 323/25 (20130101); C07C
211/27 (20130101); C07C 217/10 (20130101); C07C
217/60 (20130101); A61K 31/132 (20130101); A61P
31/12 (20180101); A61P 11/00 (20180101); C07C
217/58 (20130101); A61K 31/445 (20130101); C07D
211/46 (20130101); A61K 31/40 (20130101); A61K
31/133 (20130101); C07C 217/62 (20130101); C07C
211/35 (20130101); A61P 1/04 (20180101); C07C
211/34 (20130101); C07C 211/42 (20130101); C07C
215/60 (20130101); C07C 211/29 (20130101); C07C
215/14 (20130101); C07C 217/08 (20130101); C07C
323/47 (20130101); C07C 2602/10 (20170501); C07C
2602/42 (20170501); Y02A 50/30 (20180101); C07C
2601/08 (20170501); C07C 2601/16 (20170501); Y02A
50/402 (20180101); Y02A 50/479 (20180101); C07C
2601/14 (20170501); Y02A 50/478 (20180101); C07C
2603/74 (20170501); C07C 2601/18 (20170501); Y02A
50/401 (20180101); C07C 2601/02 (20170501) |
Current International
Class: |
A61K
31/132 (20060101); A61K 31/135 (20060101); A61K
31/44 (20060101); A61K 31/445 (20060101) |
References Cited
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669202 |
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Mar 1966 |
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BE |
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2409741 |
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Oct 2002 |
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CA |
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2007524 |
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Apr 1969 |
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FR |
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961317 |
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Jun 1964 |
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GB |
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1157143 |
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Jul 1969 |
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GB |
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1234349 |
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Jun 1971 |
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GB |
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2168986 |
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Jun 2001 |
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RU |
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803348 |
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Sep 1981 |
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SU |
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805605 |
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SU |
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WO |
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WO |
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WO 99/51213 |
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Oct 1999 |
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WO |
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WO 03/068769 |
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Aug 2003 |
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WO |
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Primary Examiner: Davis; Brian J
Attorney, Agent or Firm: Johnson & Associates
Parent Case Text
CROSS REFERENCE TO RELATED PATENT APPLICATIONS
The present application is a continuation application of U.S.
patent application Ser. No. 10/441,146 filed May 19, 2003 now
abandoned, which is a continuation-in-part application of U.S.
patent application Ser. No. 10/147,587 filed May 17, 2002 now U.S.
Pat. No. 6,951,961. The present application also claims priority to
U.S. provisional Patent Application Ser. No. 60/381,220 filed May
17, 2002.
Claims
We claim:
1. A method of treating disease caused by a mycobacterial agent,
comprising administering to a human or a non-human animal an
effective amount of a substituted ethylene diamine compound of the
formula: ##STR00080## wherein R.sub.4 is selected from H, alkyl,
aryl, alkenyl, alkynyl, aralkyl, aralkynyl, cycloalkyl,
cycloalkenyl; and wherein R.sub.1, R.sub.2 and R.sub.3 are
independently selected from H, alkenyl, alkynyl, aralkyl,
aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, halide, alkoxy,
aryloxy, alkylthio, arylthio, silyl, siloxy, amino, heteroaryl or
aryl; or wherein when R.sub.1 is selected from H, alkenyl, alkynyl,
aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, halide,
alkoxy, aryloxy, alkylthio, arylthio, silyl, siloxy, amino, and
NR.sub.2R.sub.3 is a cyclic secondary amine; or a pharmaceutically
acceptable salt thereof.
2. The method of claim 1, wherein the mycobacterial agent comprises
M. tuberculosis, M. avium-intracellulare, M. kansasii, M.
fortuitum, M. chelonae, M. leprae, M. africanum, M. bovis, M.
avium, M. microti, M. avium paratuberculosis, M. intracellulare, M.
scrofulaceum, M. xenopi, M. marinnur, or M. ulcerans.
3. The method of claim 1, wherein the disease is tuberculosis or
leprosy.
4. A method of treating disease caused by a mycobacterial agent,
comprising administering to a human or a non-human animal an
effective amount of a substituted ethylene diamine compound of the
formula ##STR00081## or a pharmaceutically acceptable salt
thereof.
5. The method of claim 4, further comprising a pharmaceutical
carrier.
6. A method for treating a disease caused by a mycobacterial agent,
comprising administering to a human or a non-human animal a
pharmaceutically effective amount of a compound of formula
##STR00082## ##STR00083## ##STR00084##
7. The method of claim 6, further comprising a pharmaceutical
carrier.
8. The method of claim 6, wherein the disease is leprosy or
tuberculosis.
9. The method of claim 1, wherein the mycobacterial agent is a drug
resistant mycobacterial strain.
10. The method of claim 6, wherein the mycobacterial agent is a
drug resistant mycobacterial strain.
11. The method of claim 1, wherein the effective amount of
substituted ethylene diamine compound is administered topically,
orally, peritoneally, subcutaneously, intramuscularly,
intraocularly, intraarterially, intravenously, or locally using an
implantable dosage unit.
12. The method of claim 1, wherein the substituted ethylene diamine
compound is administered as a solid, liquid or aerosol.
13. The method of claim 12, wherein the solid is a pill, a cream, a
soap or an implantable dosage unit.
14. The method of claim 12, wherein the liquid is a liquid
formulation adapted for injection, topical or ocular
administration.
15. The method of claim 12, wherein the aerosol comprises an
inhaler formulation.
16. The method of claim 12, wherein the solid, liquid or aerosol
comprises a sustained release matrix.
17. The method of claim 1, wherein the effective amount of
substituted ethylene diamine compound comprises from 100 to 0.1 mg
per kg of body weight.
18. The method of claim 1, wherein the effective amount of
substituted ethylene diamine compound comprises from 50 to 0.2 mg
per kg of body weight.
19. The method of claim 1, wherein the effective amount of
substituted ethylene diamine compound comprises from 25 to 0.5 mg
per kg of body weight.
20. The method of claim 1, wherein the effective amount of
substituted ethylene diamine compound comprises from 1 to 1000
mg.
21. The method of claim 4, wherein the mycobacterial agent is a
drug resistant mycobacterial strain.
22. The method of claim 4, wherein the mycobacterial agent is M.
tuberculosis, M. avium-intracellulare, M kansarii, M. fortuitum, M
chelonae, M. leprae, M africanum, M. bovis, M avium, M. microti, M.
avium paratuberculosis, M. intracellulare, M scrofulaceum, M.
xenopi, M. marinum, or M. ulcerans.
23. The method of claim 6, wherein the mycobacterial_agent is M.
tuberculosis, M. avium-intracellulare, M kansarii, M. fortuitum, M
chelonae, M. leprae, M africanum, M. bovis, M avium, M. microti, M.
avium paratuberculosis, M. intracellulare, M scrofulaceum, M.
xenopi, M. marianum, or M. ulcerans.
24. The method of claim 4, wherein the disease is leprosy or
tuberculosis.
Description
FIELD OF INVENTION
The present invention relates to methods and compositions for
treating disease caused by microorganisms, particularly
tuberculosis. The present invention also relates to methods and
compositions having improved anti-mycobacterial activity, namely
compositions comprising novel substituted ethylene diamine
compounds.
BACKGROUND OF THE INVENTION
Mycobacterial infections often manifest as diseases such as
tuberculosis. Human infections caused by mycobacteria have been
widespread since ancient times, and tuberculosis remains a leading
cause of death today. Although the incidence of the disease
declined, in parallel with advancing standards of living, since the
mid-nineteenth century, mycobacterial diseases still constitute a
leading cause of morbidity and mortality in countries with limited
medical resources. Additionally, mycobacterial diseases can cause
overwhelming, disseminated disease in immunocompromised patients.
In spite of the efforts of numerous health organizations worldwide,
the eradication of mycobacterial diseases has never been achieved,
nor is eradication imminent. Nearly one third of the world's
population is infected with mycobacterium tuberculosis complex,
commonly referred to as tuberculosis (TB), with approximately 8
million new cases, and two to three million deaths attributable to
TB yearly. Tuberculosis (TB) is the cause of the largest number of
human deaths attributable to a single etiologic agent (see Dye et
al., J. Am. Med. Association, 282, 677-686, (1999); and 2000
WHO/OMS Press Release).
After decades of decline, TB is now on the rise. In the United
States, up to 10 million individuals are believed to be infected.
Almost 28,000 new cases were reported in 1990, constituting a 9.4
percent increase over 1989. A sixteen percent increase in TB cases
was observed from 1985 to 1990. Overcrowded living conditions and
shared air spaces are especially conducive to the spread of TB,
contributing to the increase in instances that have been observed
among prison inmates, and among the homeless in larger U.S. cities.
Approximately half of all patients with "Acquired Immune Deficiency
Syndrome" (AIDS) will acquire a mycobacterial infection, with TB
being an especially devastating complication. AIDS patients are at
higher risks of developing clinical TB, and anti-TB treatment seems
to be less effective than in non-AIDS patients. Consequently, the
infection often progresses to a fatal disseminated disease.
Mycobacteria other than M. tuberculosis are increasingly found in
opportunistic infections that plague the AIDS patient. Organisms
from the M. avium-intracellulare complex (MAC), especially
serotypes four and eight, account for 68% of the mycobacterial
isolates from AIDS patients. Enormous numbers of MAC are found (up
to 10.sup.10 acid-fast bacilli per gram of tissue), and
consequently, the prognosis for the infected AIDS patient is
poor.
The World Health Organization (WHO) continues to encourage the
battle against TB, recommending prevention initiatives such as the
"Expanded Program on Immunization" (EPI), and therapeutic
compliance initiatives such as "Directly Observed Treatment
Short-Course" (DOTS). For the eradication of TB, diagnosis,
treatment, and prevention are equally important. Rapid detection of
active TB patients will lead to early treatment by which about 90%
cure is expected. Therefore, early diagnosis is critical for the
battle against TB. In addition, therapeutic compliance will ensure
not only elimination of infection, but also reduction in the
emergence of drug-resistance strains.
The emergence of drug-resistant M. tuberculosis is an extremely
disturbing phenomenon. The rate of new TB cases proven resistant to
at least one standard drug increased from 10 percent in the early
1980's to 23 percent in 1991. Compliance with therapeutic regimens,
therefore, is also a crucial component in efforts to eliminate TB
and prevent the emergence of drug resistant strains. Equally
important is the development of new therapeutic agents that are
effective as vaccines, and as treatments, for disease caused by
drug resistant strains of mycobacteria.
Although over 37 species of mycobacteria have been identified, more
than 95% of all human infections are caused by six species of
mycobacteria: M. tuberculosis, M. avium intracellulare, M.
kansasii, M. fortuitum, M. chelonae, and M. leprae. The most
prevalent mycobacterial disease in humans is tuberculosis (TB)
which is predominantly caused by mycobacterial species comprising
M. tuberculosis, M. bovis, or M. africanum (Merck Manual 1992).
Infection is typically initiated by the inhalation of infectious
particles which are able to reach the terminal pathways in lungs.
Following engulfment by alveolar macrophages, the bacilli are able
to replicate freely, with eventual destruction of the phagocytic
cells. A cascade effect ensues wherein destruction of the
phagocytic cells causes additional macrophages and lymphocytes to
migrate to the site of infection, where they too are ultimately
eliminated. The disease is further disseminated during the initial
stages by the infected macrophages which travel to local lymph
nodes, as well as into the blood stream and other tissues such as
the bone marrow, spleen, kidneys, bone and central nervous system.
(See Murray et al. Medical Microbiology, The C.V. Mosby Company
219-230 (1990)).
There is still no clear understanding of the factors which
contribute to the virulence of mycobacteria. Many investigators
have implicated lipids of the cell wall and bacterial surface as
contributors to colony morphology and virulence. Evidence suggests
that C-mycosides, on the surface of certain mycobacterial cells,
are important in facilitating survival of the organism within
macrophages. Trehalose 6,6' dimycolate, a cord factor, has been
implicated for other mycobacteria.
The interrelationship of colony morphology and virulence is
particularly pronounced in M. avium. M. avium bacilli occur in
several distinct colony forms. Bacilli which grow as transparent,
or rough, colonies on conventional laboratory media are
multiplicable within macrophages in tissue culture, are virulent
when injected into susceptible mice, and are resistant to
antibiotics. Rough or transparent bacilli, which are maintained on
laboratory culture media, often spontaneously assume an opaque R
colony morphology, at which time they are not multiplicable in
macrophages, are avirulent in mice, and are highly susceptible to
antibiotics. The differences in colony morphology between the
transparent, rough and opaque strains of M. avium are almost
certainly due to the presence of a glycolipid coating on the
surface of transparent and rough organisms which acts as a
protective capsule. This capsule, or coating, is composed primarily
of C-mycosides which apparently shield the virulent M. avium
organisms from lysosomal enzymes and antibiotics. By contrast, the
non-virulent opaque forms of M. avium have very little C-mycoside
on their surface. Both the resistance to antibiotics and the
resistance to killing by macrophages have been attributed to the
glycolipid barrier on the surface of M. avium.
Diagnosis of mycobacterial infection is confirmed by the isolation
and identification of the pathogen, although conventional diagnosis
is based on sputum smears, chest X-ray examination (CXR), and
clinical symptoms. Isolation of mycobacteria on a medium takes as
long as four to eight weeks. Species identification takes a further
two weeks. There are several other techniques for detecting
mycobacteria such as the polymerase chain reaction (PCR),
mycobacterium tuberculosis direct test, or amplified mycobacterium
tuberculosis direct test (MTD), and detection assays that utilize
radioactive labels.
One diagnostic test that is widely used for detecting infections
caused by M. tuberculosis is the tuberculin skin test. Although
numerous versions of the skin test are available, typically one of
two preparations of tuberculin antigens are used: old tuberculin
(OT), or purified protein derivative (PPD). The antigen preparation
is either injected into the skin intradermally, or is topically
applied and is then invasively transported into the skin with the
use of a multiprong inoculator (Tine test). Several problems exist
with the skin test diagnosis method. For example, the Tine test is
not generally recommended because the amount of antigen injected
into the intradermal layer cannot be accurately controlled. (See
Murray et al. Medical Microbiology, The C.V. Mosby Company 219-230
(1990)).
Although the tuberculin skin tests are widely used, they typically
require two to three days to generate results, and many times, the
results are inaccurate since false positives are sometimes seen in
subjects who have been exposed to mycobacteria, but are healthy. In
addition, instances of mis-diagnosis are frequent since a positive
result is observed not only in active TB patients, but also in
persons vaccinated with Bacille Calmette-Guerin (BCG), and those
who had been infected with mycobacteria, but have not developed the
disease. It is hard therefore, to distinguish active TB patients
from the others, such as household TB contacts, by the tuberculin
skin test. Additionally, the tuberculin test often produces a
cross-reaction in those individuals who were infected with
mycobacteria other than M. tuberculosis (MOTT). Therefore,
diagnosis using the skin tests currently available is frequently
subject to error and inaccuracies.
The standard treatment for tuberculosis caused by drug-sensitive
organisms is a six-month regimen consisting of four drugs given for
two months, followed by two drugs given for four months. The two
most important drugs, given throughout the six-month course of
therapy, are isoniazid and rifampin. Although the regimen is
relatively simple, its administration is quite complicated. Daily
ingestion of eight or nine pills is often required during the first
phase of therapy; a daunting and confusing prospect. Even severely
ill patients are often symptom free within a few weeks, and nearly
all appear to be cured within a few months. If the treatment is not
continued to completion, however, the patient may experience a
relapse, and the relapse rate for patients who do not continue
treatment to completion is high. A variety of forms of
patient-centered care are used to promote adherence with therapy.
The most effective way of ensuring that patients are taking their
medication is to use directly observed therapy, which involves
having a member of the health care team observe the patient take
each dose of each drug. Directly observed therapy can be provided
in the clinic, the patient's residence, or any mutually agreed upon
site. Nearly all patients who have tuberculosis caused by
drug-sensitive organisms, and who complete therapy will be cured,
and the risk of relapse is very low ("Ending Neglect: The
Elimination of Tuberculosis in the United States" ed. L. Geiter
Commnittee on the Elimination of Tuberculosis in the United States
Division of Health Promotion and Disease Prevention, Institute of
Medicine. Unpublished.)
What is needed are effective therapeutic regimens that include
improved vaccination and treatment protocols. Currently available
therapeutics are no longer consistently effective as a result of
the problems with treatment compliance, and these compliance
problems contribute to the development of drug resistant
mycobacterial strains.
Ethambutol (EMB) is a widely used antibiotic for the treatment of
TB, with over 300 million doses delivered for tuberculosis therapy
in 1988.
##STR00001##
Ethambutol, developed by Lederle Laboratories in the 1950s, has low
toxicity and is a good pharmacokinetic. However, ethambutol has a
relatively high Minimum Inhibition Concentration (MIC) of about 5
.mu.g/ml, and can cause optic neuritis. Thus, there is an
increasing need for new, and more effective, therapeutic
compositions (See for example, U.S. Pat. Nos. 3,176,040, 4,262,122;
4,006,234; 3,931,157; 3,931,152; U.S. Re. 29,358; and Hausler et
al., Bioorganic & Medicinal Chemistry Letters 11 (2001)
1679-1681). In the decoder years since the discovery of the
beneficial effects of ethambutol, few pharmacological advances in
TB treatment have been developed. Moreover, with the combined
emergence of drug resistant strains, and the more prevalent spread
of mycobacterial disease, it is becoming seriously apparent that
new therapeutic compositions are crucial in the fight against
tuberculosis.
Clearly effective therapeutic regimens that include improved
vaccination and treatment protocols are needed. A therapeutic
vaccine that would prevent the onset of tuberculosis, and therefore
eliminate the need for therapy is desirable. Although currently
available therapeutics such as ethambutol are effective, the
emergence of drug resistant strains has necessitated new
formulations and compositions that are more versatile than
ethambutol. Currently available therapeutics are no longer
consistently effective as a result of the problems with treatment
compliance, lending to the development of drug resistant
mycobacterial strains. What is needed are new anti-tubercular drugs
that provide highly effective treatment, and shorten or simplify
tuberculosis chemotherapy.
SUMMARY OF THE INVENTION
The present invention comprises methods and compositions comprising
ethylene diamine compounds effective for the treatment of
infectious disease. The present invention also provides methods and
compositions comprising substituted ethylene diamines having
improved anti-mycobacterial activity, including substituted
ethylene diamines having improved anti-tuberculosis activity.
The present invention contemplates substituted ethylene diamines,
which can derive from a variety of amine compounds. In the present
invention, the substituted ethylene diamines are based on the
following structure.
##STR00002##
The substituted ethylene diamine compounds described herein are
synthesized and screened for activity as follows. A chemical
library of substituted ethylene diamines is prepared on a solid
polystyrene support using split and pool technologies. This
technique allows for the synthesis of a diverse set of substituted
ethylene diamines. These diamines are screened for anti-TB activity
using in vitro, biological assays, including a High-Throughput
Screening (HTS) assay, based on the recently completed genomic
sequence of M. tuberculosis, and a Minimum Inhibition-Concentration
(MIC) assay.
The methods and compositions described herein comprise substituted
ethylene diamines that are effective against disease caused by
infectious organisms, including, but not limited to, bacteria and
viruses. One embodiment of the invention provides methods and
compositions comprising substituted ethylene diamines that are
effective against mycobacterial disease. Another embodiment of the
invention provides methods and compositions comprising substituted
ethylene diamines that have MIC of 50 .mu.M or lower for
mycobacterial disease. Another embodiment of the present invention
comprises substituted ethylene diamines that have an MIC of 25
.mu.M or lower for mycobacterial disease. Yet another embodiment of
the present invention comprises substituted ethylene diamines that
have an MIC of 12.5 .mu.M or lower for mycobacterial disease.
Another embodiment of the present invention comprises substituted
ethylene diamines that have an MIC of 5 .mu.M or lower for
mycobacterial disease In another embodiment of the present
invention, the methods and compositions comprise substituted
ethylene diamines with HTS Luc activity of 10% or greater. In yet
another embodiment of the present invention, the methods and
compositions comprise substituted ethylene diamines, wherein one
amine group is derived from a primary amine, and wherein the other
amine group is derived from a primary or secondary amine. In
another embodiment of the present invention, the methods and
compositions comprise substituted ethylene diamines, wherein one
amine is derived from cis-(-)myrtanylamine, cyclooctylamine,
2,2-diphenylethylamine, 3,3-diphenylpropylamine, (+)-bomylamine,
1-adamantanemethylamine, (+)-isopinocampheylamine; or
(-)-isopinocampheylamine.
The present invention contemplates various salt complexes and other
substituted derivatives of the substituted ethylene diamines. The
present invention also contemplates enantiomers and other
stereoisomers of the substituted ethylene diamines and their
substituted derivatives. The present invention further contemplates
treatment for animals, including, but not limited to, humans.
Accordingly, it is an object of the present invention to provide
methods and compositions for the treatment and prevention of
diseases caused by microorganisms.
Accordingly, it is an object of the present invention to provide
methods and compositions for the treatment and prevention of
infectious diseases.
Another object of the present invention is to provide methods and
compositions for the treatment and prevention of mycobacterial
disease, including but not limited to, tuberculosis.
Yet another object of the present invention is to provide methods
and compositions for the treatment and prevention of infectious
diseases using compositions comprising substituted ethylene
diamines.
Another object of the present invention is to provide methods and
compositions for the treatment and prevention of mycobacterial
disease using compositions comprising substituted ethylene
diamines.
Still another object of the present invention is to provide methods
and compositions for the treatment and prevention of tuberculosis
using compositions comprising substituted ethylene diamines.
Another object of the present invention is to provide methods and
compositions for the treatment and prevention of tuberculosis using
compositions comprising substituted ethylene diamines, wherein the
diamine has an MIC of 50 .mu.M, or less.
Another object of the present invention is to provide methods and
compositions for the treatment and prevention of tuberculosis using
compositions comprising substituted ethylene diamines, wherein the
diamine has an MIC of 25 .mu.M, or less.
Another object of the present invention is to provide methods and
compositions for the treatment and prevention of tuberculosis using
compositions comprising substituted ethylene diamines, wherein the
diamine has an MIC of 12.5 .mu.M, or less.
Yet another object of the present invention is to provide methods
and compositions for the treatment and prevention of tuberculosis
using compositions comprising substituted ethylene diamines,
wherein the diamine has an MIC of 5 .mu.M, or less.
Yet another object of the present invention is to provide methods
and compositions for the treatment and prevention of tuberculosis
using compositions comprising substituted ethylene diamines,
wherein the diamine has HTS/Luc activity of 10% or greater.
Another object of the present invention is to provide methods and
compositions for the treatment and prevention of tuberculosis using
compositions comprising substituted ethylene diamines, wherein one
amine group is derived from a primary amine, and the other amine
group is derived from a primary or secondary amine.
Yet another object of the present invention is to provide methods
and compositions for the treatment and/or prevention of
tuberculosis using compositions comprising substituted ethylene
diamines, wherein one amine is derived from cis-(-)myrtanylamine,
cyclooctylamine, 2,2-diphenylethylamine, 3,3-diphenylpropylamine,
(+)-bornylamine, 1-adamantanemethylamine, (+)-isopinocampheylamine;
or (-)-isopinocampheylamine.
Yet another object of the present invention is to provide
composition for the therapeutic formulation for the treatment and
prevention of mycobacterial disease.
Another object of the present invention is to provide compositions
for therapeutic formulations for the treatment and prevention of
mycobacterial disease caused by mycobacterial species comprising M.
tuberculosis complex, M. avium intracellulare, M. kansarii, M.
fortuitum, M. chelonoe, M. leprae, M. africanum, M. microti, or M.
bovis.
These and other objects, features and advantages of the present
invention will become apparent after a review of the following
detailed description of the disclosed embodiments and the appended
claims.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 represents a flow chart schematic showing various solid
support syntheses used to prepare substituted ethylene
diamines.
FIGS. 2(a)-2(ac) provide chemical structures of a variety of
primary amines.
FIGS. 3(a)-3(f) provide chemical structures of a variety of acyclic
secondary amines.
FIGS. 4(a)-4(i) provide chemical structures of a variety of cyclic
secondary amines.
FIG. 5 represents a flow schematic for a representative reaction
pool of ten substituted ethylene diamines.
FIG. 6 is a graph of Luminescence Count per Second (LCPS) versus
concentration showing HTS Luc assay results for pooled substituted
ethylene diamine compounds.
FIG. 7 is a graph of LCPS versus concentration showing HTS Luc
assay results for individual substituted ethylene diamine
compounds.
FIG. 8 is a graph of LCPS versus concentration showing HTS Luc
assay results for individual substituted ethylene diamine
compounds.
FIG. 9 is a bar graph providing a summary of MIC activities for
discrete substituted ethylene diamines.
FIG. 10 is a bar graph providing a summary of Luciferase activity
of discrete substituted ethylene diamines with at least 10%
activity in reference to ethambutol at 3.1 .mu.M.
FIG. 11 is a bar graph showing the frequency of occurrences of the
selected amine monomers in the substituted ethylene diamine
compounds that were active against TB. Amine monomers are
represented by their numerical designations.
FIG. 12 represents a flow schematic showing a synthesis of
N-Geranyl-N'-(2-adamanthyl)ethane-1,2-diamine (compound 109).
FIG. 13 is a flow schematic showing a synthesis of
N-(Cyclooctyl)-N'-(1R,2R,3R,5S)-(-)-isopinocampheylethane-1,2-diamine
as hydrochloride (compound 59).
FIG. 14 is a mass spec profile for one representative sample well
containing pooled substituted ethylene diamine compounds.
FIG. 15 is a mass spec profile for compound 109,
N-Geranyl-N.sup.1-(2-adamanthyl) ethane-1,2-diamine.
FIG. 16 is a proton NMR profile for compound 109,
N-Geranyl-N.sup.1-(2-adamanthyl) ethane-1,2-diamine.
FIG. 17 is a bar graph of data from a Colony Forming Units/Lung
(CFUI/Lung) study showing CFU/Lung growth over time in days for
various compounds.
FIG. 18 is a bar graph of data from a CFU/Lung study showing
CFU/Lung growth over time in days for various compounds.
FIG. 19 is a bar graph of data from a CFU/Lung study showing
CFU/Lung growth over time in days for various compounds.
FIG. 20 is a bar graph of data from a lesion study showing visible
lesions over time after treatment with various compounds.
FIG. 21 provides a schematic demonstrating the identification of a
drug candidate FIG. 22 provides the compounds tested for in vivo
efficacy.
FIG. 23 is a graph showing the results of in vivo studies of
compounds 73 and 109 at 1 and 10 mg/g doses (spleen).
FIG. 24 is a graph showing the results of in vivo studies of
compounds 73 and 109 at 1 and 10 mg/kg doses (lungs).
FIG. 25 is a graph showing in vivo studies of compounds 59 and 111
at 1 and 10 mg/kg doses (spleen).
FIG. 26 is a graph showing in vivo studies of compounds 59 and 111
at 1 and 10 mg/kg doses (lungs).
FIG. 27 is a graph showing the results of efficacy testing of the
compounds 58, 73, 109, and 111 in C57BL.6 mice infected with M.
tuberculosis H37Rv (spleen). Mice were infected i.v. with
5.times.10.sup.6 CFU M. tuberculosis H37Rv; treatment with drugs
started 18 days following infection. EC-EC--early control, CFU in
lungs of mice at the day of chemotherapy start. Mice received:
1--untreated mice, 2--INH (25 mg/kg), 3--EMB (100 mg/kg), 4--comp.
109 (25 mg/kg), 4*--comp. 109 (10 mg/kg), 4**--comp. 109 (0.1
mg/kg), 5--comp. 58 (25 mg/kg), 6--comp. 73 (25 mg/kg), 7--comp.
111 (25 mg/kg).
FIG. 28 is a graph showing the results of efficacy testing of the
compounds 58, 73, 109, and 111 in C57BL.6 mice infected with M.
tuberculosis H37Rv (lungs). Mice were infected i.v. with
5.times.10.sup.6 CFU M. tuberculosis H37Rv; treatment with drugs
started 18 days following infection. EC-EC--early control, CFU in
lungs of mice at the day of chemotherapy start. Mice received:
1--untreated mice, 2--H (25 mg/kg), 3--EMB (100 mg/kg), 4--comp.
109 (25 mg/kg), 4*--comp. 109 (10 mg/kg), 4**--comp. 109 (0.1
mg/kg), 5--comp. 58 (25 mg/kg), 6--comp. 73 (25 mg/kg), 7--comp.
111 (25 mg/kg).
FIG. 29 provides LC/MS data of tested compounds.
FIG. 30 provides a graph showing results of PK studies with a
cassette dosing of tested compounds to mice. Oral delivery.
Compound NSC 722039 in the study reads as the compound 37, NSC
722040--compound 59, NSC 722041--compound 109.
FIG. 31 provides a graph showing results of PK studies with a
cassette dosing of tested compounds to mice. Peritoneal delivery.
Compound NSC 722039 in the study reads as the compound 37, NSC
722040--compound 59, NSC 722041--compound 109.
FIG. 32 provides a graph showing results of PK studies with a
cassette dosing of tested compounds to mice. Intravenous delivery.
Compound NSC 722039 in the study reads as the compound 37, NSC
722040--compound 59, NSC 722041--compound 109.
FIG. 33 provides a graph showing the results of PK Studies of the
compound 109 in mice.
FIG. 34. Tissue distribution of 109 in mice (i.v., 3 mg/kg).
FIG. 35. Tissue distribution of 109 in mice p.o., 25 mg/kg).
FIG. 36 Metabolism of the compound 109 in mouse urine.
FIG. 37. No glucoronidation metabolites of 109 were found in mouse
urine.
FIG. 41. Scheme 1. Synthesis of 100,000 compound library of
ethambutol analogues on solid support.
FIG. 41. Scheme 2. Attempts to synthesize SQBisAd on solid
support.
FIG. 42 provides structures of representative targeted diamines
prepared via acylation by amino acids.
FIG. 43 provides Table 25 summarizing data for synthesized plates
of diamines for the prepared library of targeted 20,000 ethambutol
analogs.
FIG. 44 provides Scheme 5 showing the synthesis of the diamine
library using amino acids as linkers.
FIG. 45 provides a schematic showing the occurrence of amine
monomers in the hits that were generated in the original 100,000
compound library of EMB analogs.
FIG. 46 provides a schematic showing structural diversity among
primary amines.
FIG. 47 provides Table 26 listing the amino acids that were used in
the prepartion of the diamine library.
FIG. 48 provides carbonyl compounds used as reagents in the
synthesis of the diamine library.
FIG. 49 provides Table 27 showing carbonyl compounds used in the
masterplate for the synthesis of the diamine library.
FIG. 50 provides representative examples of MIC and Lux data for
the diamine library.
FIG. 51 provides a schematic showing the occurrence of alkylating
monomers in final diamine products with anti-TB activity.
FIG. 52 provides the layout of a representative 96-well
deconvolution plate.
FIG. 53 provides a list of compound hits and structures for the
modified linker diamine library.
DETAILED DESCRIPTION
The present invention may be understood more readily by reference
to the following detailed description of the specific embodiments
included herein. However, although the present invention has been
described with reference to specific details of certain embodiments
thereof, it is not intended that such details should be regarded as
limitations upon the scope of the invention. The entire text of the
references mentioned herein are hereby incorporated in their
entireties by reference including U.S. Patent Application Ser. No.
10/147,587 filed May 17, 2002, and U.S. Provisional Patent
Application Ser. No. 60/381,220 filed May 17, 2002.
Mycobacterial infections, such as those causing tuberculosis, once
thought to be declining in occurrence, have rebounded, and again
constitute a serious health threat. Tuberculosis (TB) is the cause
of the largest number of human deaths attributed to a single
etiologic agent with two to three million people infected with
tuberculosis dying each year. Areas where humans are crowded
together, or living in substandard housing, are increasingly found
to have persons affected with mycobacteria. Individuals who are
immunocompromised are at great risk of being infected with
mycobacteria and dying from such infection. In addition, the
emergence of drug-resistant strains of mycobacteria has led to
treatment problems of such infected persons.
Many people who are infected with mycobacteria are poor, or live in
areas with inadequate healthcare facilities. As a result of various
obstacles (economical, education levels, etc.), many of these
individuals are unable to comply with the prescribed therapeutic
regimens. Ultimately, persistent non-compliance by these and other
individuals results in the prevalence of disease. This
noncompliance is frequently compounded by the emergence of
drug-resistant strains of mycobacteria. Effective compositions and
vaccines that target various strains of mycobacteria are necessary
to bring the increasing number of tuberculosis cases under
control.
Chemotherapy is a standard treatment for tuberculosis. Some current
chemotherapy treatments require the use of three or four drugs, in
combination, administered daily for two months, or administered
biweekly for four to twelve months. Table 1 lists several treatment
schedules for standard tuberculosis drug regimens.
TABLE-US-00001 TABLE 1 Treatment Schedules for Standard TB Drug
Regimens. INDUCTION STANDARD PHASE CONTINUATION DRUG Dosing PHASE
REGIMEN Schedule DURATION DRUG Dosing Schedule DURATION Isoniazid
Daily, DOT 8 weeks Isoniazid 2/week, DOT 16 weeks Rifampicin Daily,
DOT 8 weeks Rifampicn 2/week, DOT 16 weeks Pyrazinamide Daily, DOT
8 weeks Ethambutol or Daily, DOT 8 weeks Streptomycin
Decades of misuse of existing antibiotics and poor compliance with
prolong and complex therapeutic regimens has led to mutations of
the mycobacterium tuberculosis and has created an epidemic of drug
resistance that threatens tuberculosis control world wide. The vast
majority of currently prescribed drugs, including the front line
drugs, such as isoniazid, rifampin, pyrazinamide, ethambutol and
streptomycin were developed from the 1950s to the 1970s. Thus, this
earlier development of tuberculosis chemotherapy did not have at
its disposal the implications of the genome sequence of
Mycobacterium tuberculosis, the revolution in pharmaceutical drug
discovery of the last decades, and the use of national drug testing
and combinational chemistry.
Consequently, the treatments of drug-resistant M. tuberculosis
strains, and latent tuberculosis infections, require new
anti-tuberculosis drugs that provide highly effective treatments,
and shortened and simplified tuberculosis chemotherapies. Moreover,
it is desirable that these drugs be prepared by a low-cost
synthesis, since the demographics of the disease dictate that cost
is a significant factor.
The present invention provides methods and compositions comprising
a class of substituted ethylene diamine compounds effective in
treatment and prevention of disease caused by microorganisms
including, but not limited to, bacteria. In particular, the methods
and compositions of the present invention are effective in
inhibiting the growth of the microorganism, M. tuberculosis. The
methods and compositions of the present invention are intended for
the treatment of mycobacteria infections in human, as well as other
animals. For example, the present invention may be particularly
useful for the treatment of cows infected by M. bovis.
As used herein, the term "tuberculosis" comprises disease states
usually associated with infections caused by mycobacteria species
comprising M. tuberculosis complex. The term "tuberculosis" is also
associated with mycobacterial infections caused by mycobacteria
other than M. tuberculosis (MOTT). Other mycobacterial species
include M. avium-intracellulare, M. kansarii, M. fortuitum, M.
chelonae, M. leprae, M. africanum, and M. microti, M. avium
paratuberculosis, M. intracellulare, M. scrofulaceum, M. xenopi, M.
marinum, M. ulcerans.
The present invention further comprises methods and compositions
effective for the treatment of infectious disease, including but
not limited to those caused by bacterial, mycological, parasitic,
and viral agents. Examples of such infectious agents include the
following: staphylococcus, streptococcaceae, neisseriaaceae, cocci,
enterobacteriaceae, pseudomonadaceae, vibrionaceae, campylobacter,
pasteurellaceae, bordetella, francisella, brucella, legionellaceae,
bacteroidaceae, gram-negative bacilli, clostridium,
corynebacterium, propionibacterium, gram-positive bacilli, anthrax,
actinomyces, nocardia, mycobacterium, treponema, borrelia,
leptospira , mycoplasma, ureaplasma, rickettsia, chlamydiae,
systemic mycoses, opportunistic mycoses, protozoa, nematodes,
trematodes, cestodes, adenoviruses, herpesviruses, poxviruses,
papovaviruses, hepatitis viruses, orthomyxoviruses,
paramyxoviruses, coronaviruses, picornaviruses, reoviruses,
togaviruses, flaviviruses, bunyaviridae, rhabdoviruses, human
immunodeficiency virus and retroviruses.
The present invention further provides methods and compositions
useful for the treatment of infectious disease, including by not
limited to, tuberculosis, leprosy, Crohn's Disease, aquired
immunodeficiency syndrome, lyme disease, cat-scratch disease, Rocky
Mountain Spotted Fever and influenza.
The anti-infective methods and compositions of the present
invention contain one or more substituted ethylene diamine
compounds. In particular, these compounds encompass a wide range of
substituted ethylene diamine compounds having the following general
formula:
##STR00003## where "R.sub.1NH" is typically derived from a primary
amine, and "R.sub.2R.sub.3N" is typically derived from a primary or
secondary amine. The ethylene diamines of the present invention are
prepared by a modular approach using primary and secondary amines
as building blocks, and coupling the amine moieties with an
ethylene linker building block. Representative primary amines,
acyclic secondary amines, and cyclic secondary amines are shown in
FIGS. 2, 3, and 4, respectively.
Generally, chemical moieties R.sub.1, R.sub.2, and R.sub.3 of the
ethylene diamine compounds of the present invention are
independently selected from H, alkyl; aryl; alkenyl; alkynyl;
aralkyl; aralkenyl; aralkynyl; cycloalkyl; cycloalkenyl;
heteroalkyl; heteroaryl; halide; alkoxy; aryloxy; alkylthio;
arylthio; silyl; siloxy; a disulfide group; a urea group; amino;
and the like, including straight or branched chain derivatives
thereof, cyclic derivatives thereof, substituted derivatives
thereof, heteroatom derivatives thereof, heterocyclic derivatives
thereof, functionalized derivatives thereof, salts thereof, such
salts including, but not limited to hydrochlorides and acetates,
isomers thereof, or combinations thereof. For example,
nitrogen-containing heterocyclic moieties include, but are not
limited to, groups such as pyridinyl (derived from pyridine, and
bonded through a ring carbon), piperidinyl (derived from piperidine
and bonded through the ring nitrogen atom or a ring carbon), and
pyrrolidinyl (derived from pyrrolidine and bonded through the ring
nitrogen atom or a ring carbon). Examples of substituted, or
functionalized, derivatives of R.sub.1, R.sub.2, and R.sub.3
include, but are not limited to, moieties containing substituents
such as acyl, formyl, hydroxy, acyl halide, amide, amino, azido,
acid, alkoxy, aryloxy, halide, carbonyl, ether, ester, thioether,
thioester, nitrile, alkylthio, arythio, sulfonic acid and salts
thereof, thiol, alkenyl, alkynyl, nitro, imine, imide, alkyl, aryl,
combinations thereof, and the like. Moreover, in the case of
alkylated derivatives of the recited moieties, the alkyl
substituent may be pendant to the recited chemical moiety, or used
for bonding to the amine nitrogen through the alkyl
substituent.
Examples of chemical moieties R.sub.1, R.sub.2, and R.sub.3 of the
present invention include, but are not limited to: H; methyl;
ethyl; propyl; butyl; pentyl; hexyl; heptyl; octyl; ethenyl;
propenyl; butenyl; ethynyl; propynyl; butynyl; cyclopropyl;
cyclobutyl; cyclopentyl; cyclohexyl; cyclooctyl cyclobutenyl;
cyclopentenyl; cyclohexenyl; phenyl; tolyl; xylyl; benzyl;
naphthyl; pyridinyl; furanyl; tetrahydro-1-napthyl; piperidinyl;
indolyl; indolinyl; pyrrolidinyl; 2-(methoxymethyl) pyrrolidinyl;
piperazinyl; quinolinyl; quinolyl; alkylated-1,3-dioxolane;
triazinyl; morpholinyl; phenyl pyrazolyl; indanyl; indonyl;
pyrazolyl; thiadiazolyl; rhodaninyl; thiolactonyl; dibenzofuranyl;
benzothiazolyl; homopiperidinyl; thiazolyl; quinonuclidinyl;
isoxazolidinonyl; any isomers, derivatives, or substituted analogs
thereof; or any substituted or unsubsfituted chemical species such
as alcohol, ether, thiol, thioether, tertiary amine, secondary
amine, primary amine, ester, thioester, carboxylic acid, diol,
diester, acrylic acid, acrylic ester, methionine ethyl ester,
benzyl-1-cysteine ethyl ester, imine, aldehyde, ketone, amide, or
diene. Further examples of chemical moieties R.sub.1, R.sub.2, and
R.sub.3 of the present invention include, but are not limited to,
the following species or substituted or alkylated derivatives of
the following species, covalently bonded to the amine nitrogen:
furan; tetrahydrofuran; indole; piperazine; pyrrolidine;
pyrrolidinone; pyridine; quinoline; anthracene;
tetrahydroquinoline; naphthalene; pyrazole; imidazole; thiophene;
pyrrolidine; morpholine; and the like. One feature of the recited
species or substituted or alkylated derivatives of these species,
is that they may be covalently bonded to the amine nitrogen in any
fashion, including through the pendant substituent or alkyl group,
through the heteroatom as appropriate, or through a ring atom as
appropriate, as understood by one of ordinary skill in the art.
The chemical moieties R.sub.1, R.sub.2, and R.sub.3 of the present
invention also include, but are not limited to, cyclic alkanes and
cyclic alkenes, and include bridged and non-bridged rings. Examples
of bridged rings include, but are not limited to, the following
groups: isopinocamphenyl; bomyl; norbornyl; adamantanetetyl;
cis-(-)myrtanyl; adamantyl; noradamantyl;
6-azabicyclo[3.2.1]octane; exo-norbornane; and the like.
In one embodiment of the present invention, NR.sub.2R.sub.3 is
derived from a cyclic secondary amine. Examples of a cyclic
chemical moiety, NR.sub.2R.sub.3, of the present invention include,
but are not limited to, 4-benzyl-piperidine; 3-piperidinemethanol;
piperidine; tryptamine; moropholine; 4-piperidinopiperidine; ethyl
1-piperazine carboxylate; 1-(2-amino-ethyl)-piperazine;
decahydroquinoline; 1,2,3,4-tetrahydro-pyridoindole (reaction at
either amine); 3-amino-5-phenyl pyrazole; 3-aminopyrazole;
1-(2-fluorophenyl) piperazine; 1-proline methyl ester; histidinol;
1-piperonyl-piperazine; hexamethyleimine; 4-hydroxypiperidine;
2-piperidinemethanol; 1,3,3-trimethyl-6-azabicyclo[3.2.1]octane;
3-pyrrolidinol; 1-methylpiperazine; (S)-(+)-(2-pyrolidinylmethyl)
pyrrolidine; 1-methylhomopiperazine; 2-ethyl-piperidine; 1, 2, 3,
4-tetrahydroisoquinoline; 1-(4-fluorophenyl) piperazine;
d,1-tryptophan methyl ester; tert-butyl
(15,45)-(-)-2,5-diazabiclyclo[2.2.1]heptane-2-carboxylate;
isonipecotamide; heptamethyleneimine; alpha-methyltryptamine;
6,7-dimethoxy-1, 2, 3, 4-tetrahydroisoquinoline;
3-aminopyrrolidine; 3,5-dimethylpiperidine; 2,6-dimethylmorpholine;
1,4-dioxo-8-azaspiro[4.5]decane;
1-methol-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline;
1,3,4,6,7,8-hexahydro-2H-pyrido (1,2-A) pyrimidine;
1,2,3,4-tetrahydroquinoline; 1-(2-methoxyphenyl) piperazine;
1-(2-(2-hydroxyethoxy)ethyl) piperazine; (S)-(+)-2-(aminomethyl)
pyrroli-dine; (3S(3a, 4Ab),
8Ab)-N-t-butyl-D-ecahydro-3-isoquino-linecarboxamide;
(R)-cycloserine; homopiperazine; 2,6-dimethylpiperazine (reaction
at either amine); iminodibenzyl; 5-methoxytryptamine;
4,4'-bipiperidine; 1-(2-hydroxyethyl) piperazine;
4-methylpiperidine; 1-histidine methyl ester; or methyl
pipecoliate.
The R.sub.1HN substituent is derived from a primary amine. The
R.sub.2R.sub.3N substituent is typically derived from a primary or
secondary amine, but may also arise from an amino acid, or an amino
acid precursor. The amino acid can transform into an amino alcohol.
When an amino acid is employed as the source of the R.sub.2R.sub.3N
moiety, the precursor compound may be selected from, among others,
the following compounds and their derivatives: d,1-tryptophan
methyl ester; 1-methionine ethyl ester; 1-lysine methyl ester (via
reaction at either primary amine); (S)-benzyl-1-cysteine ethyl
ester; 1-arginine methyl ester (via reaction at either primary
amine); 1-glutamic acid ethyl ester; 1-histidine methyl ester; or
(3S (3a, 4Ab), 8A b)-N-t-butyl-D-ecahydro-3-isoquino
linecarboxamide.
The R.sub.4 moiety of the substituted ethylene diamine compounds of
the present invention is typically selected from H, alkyl or aryl,
but R.sub.4 can also constitute alkenyl, alkynyl, aralkyl,
aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, and the like.
Examples of the R.sub.4 chemical moiety include, but are not
limited to: H; methyl; ethyl; propyl; butyl; pentyl; hexyl; heptyl;
octyl; ethenyl; propenyl; butenyl; ethynyl; propynyl; butynyl;
cyclobutyl; cyclopentyl; cyclohexyl; cyclobutenyl; cyclopentenyl;
cyclohexenyl; phenyl; tolyl; xylyl; benzyl; naphthyl; straight or
branched chain derivatives thereof; cyclic derivatives thereof;
substituted, functionalized, and heteroatom derivatives thereof;
and heterocyclic derivatives thereof, and the like. Typically,
R.sub.4 is selected from H, methyl, ethyl, butyl or phenyl.
However, when R.sub.4 is "H" the ethylene diamine does not contain
ethambutol.
A majority of the ethylene diamine compounds described hrein are
preferably prepared using a solid support synthesis, as set forth
in one of the representative reaction schemes shown in FIG. 1.
However, when R.sub.4 is H, the reaction does not proceed well when
sterically hindered amines are used for R.sub.1NH.sub.2, or when
diamines, such as amino alkylenemorpholine, or
aminoalkylene-piperidines, are used for R.sub.1NH.sub.2. When
R.sub.4 is methyl, or phenyl, sterically hindered amines used for
R.sub.3R.sub.2NH do not work well due to steric hindrance at the
reaction site. In this case, a competing hydrolysis reaction
producing the corresponding amino alcohols, and incomplete
reduction of the amidoethyleneamines, interfere with the reaction
scheme. As a result, the desired diamine products form in low
yields.
The preparation of the ethylene diamines is preferably accomplished
in six steps, using a rink-acid resin. The first step of the
synthesis is converting the rink-acid resin to rink-chloride by
treatment with triphenylphosphine and hexachloroethane in
tetrahydrofuran (THF). This step is followed by addition of the
primary amine in the presence of Hunig's base (EtN(i-Pr).sub.2) in
dichloroethane. The third step is the acylation of the
resin-attached amine using either one of the two acylation routes
shown in FIG. 1. The acylation step is preferably accomplished
using either ahloroacetyl chloride, .alpha.-bromo-.alpha.-methyl
acetylbromide, .alpha.-bromo-.alpha.-ethylacetyl bromide,
.alpha.-bromo-.alpha.-butyl acetylbromide, or
.alpha.-chloro-.alpha.-phenyl-acetylchloride, each in the presence
of pyridine in THF. Other acylation reagents known to those skilled
in the art may also be used, however, the .alpha.-bromoacetyl
halides result in low product yields, which may be attributed to
HBr elimination. The acylation may also be accomplished via a
peptide coupling mechanism using .alpha.-bromo-.alpha.-methylacetic
acid, or .alpha.-chloro-.alpha.-methylacetic acid, in the presence
of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBrop) and NIN-diisopropylethyl amine
(EtN(i-Pr).sub.2) in dichloromethane (DCM) and dimethylformamide
(DMF). Again, other acylation reagents known to those skilled in
the art may also be used. The acylation step is preferably
performed twice to achieve better acylated product yields.
Introduction of the second nitrogen moiety is preferably achieved
in the presence of Hunig's base in dimethylformamide (DMF).
Reduction of the intermediate amine-amide is carried out using
Red-A1 (3.4M solution of sodium bis (2-methoxyethoxy) aluminum
hydride in toluene). The final product is cleaved from the resin
support using a 10% solution (by volume) of trifluoroacetic acid
(TFA) in dichloromethane (DCM). The solvent is evaporated, and the
TFA salts of the final diamine products are analyzed by mass spec,
and screened against M. tuberculosis for effectiveness. Some of the
substituted ethylene diamines, prepared using the above-described
solid-support synthesis, are also prepared using a solution phase
synthesis described below.
Formation of the Substituted Ethylene Diamine Library
The solid support syntheses, shown in FIG. 1, are preferably used
to prepare a substituted ethylene diamine library. Solid phase
synthesis offers at least three principal advantages: (i) a reduced
need for chromatographic procedures, (ii) the use of excess
reagents to drive a reaction forward in high yields, and (iii) the
use of split and pool technologies for the synthesis of a large
number of compounds. Solid support syntheses of 1,2-diamine
libraries have previously been accomplished by the reduction of
short peptides (Cuervo et al., Peptides 1994: Proceedings of the
European Peptide Symposium; Maia HSL Ed., Esom: Leiden, 1995,
465-466). However, as described herein, an ethylene diamine library
is created using amines, rather than simple amino acids, to allow
for greater diversity in the building-block monomers. The first
three steps of each support synthesis: the activation of the
Rink-acid resin, the addition of the first amine, and the acylation
step are carried out in 10 ml tubes on a QUEST.RTM. 210 Synthesizer
manufactured by ARGONAUT TECHNOLOGIES.RTM., Inc., Foster City,
Calif. The synthesizer handles up to twenty simultaneous reactions
in 5 ml or 10 ml reaction vessels to allow for rapid synthesis of
target compounds. The synthesizer provides programmable temperature
control and agitation, and the automated delivery of solvents into
the reaction vessels. The addition of the second amine, the
reduction with Red-A1, and the cleavage from the solid support are
carried out in 2 ml wells in a 96-well, chemically resistant
plate.
Prior to the solid support synthesis, each amine, within numbers 1
to 288, as shown in FIGS. 2, 3, and 4, is dissolved in DMF as a one
molar solution, and organized in three, 96-well plates (one amine
per well), to yield three master plates of these amines. An
individual haloacetyl amide from each primary amine and a
particular R group, is formed in the first three steps of the
support synthesis. Individual haloacetyl amides are then pooled
into groups of ten or thirty. A suspension of the pooled resins in
a 2:1 mixture of DCM/THF is evenly distributed into one, two or
three reaction plates to assure 15-20 mg of the suspension per
well. The number of reaction plates used is based on the amount of
suspension available. Each well of pooled resins is reacted with a
corresponding amine from the master plates. FIG. 5 provides a flow
schematic for a representative pool. Each reaction occurs in a
separate well, in the presence of Hunig's base in DMF at
70-75.degree. C. for 16-20 hours. Each resulting amine-amide is
reduced using 65+w % Red-A1 at room temperature. The reduction is
followed by cleavage with 10% vol. TFA in DCM. The solvents in each
reaction well are evaporated, and the TFA salts of the diamines
analyzed (mass spec), and screened against M. tuberculosis. One
plate of pooled diamines are screened against M. smegmatis. Two
randomly selected rows in each plate; i.e., 24 samples per 96-well
plate, or 25% of the library, are examined by mass spectroscopy.
Specific protocols and detailed methods are provided below in the
Examples.
Screening Against M. tuberculosis
An entire library of synthesized substituted ethylene diamines
(targeted number of compounds about 100,000), prepared as described
above, was screened, in vitro, against M. tuberculosis in
ethambutol (EMB) sensitive Luc-assay. The MIC (Minimum Inhibition
Concentration) was also determined. The MIC is the minimum
concentration of a growth inhibitor, here the substituted ethylene
diamine, where there is no multiplication of the microorganism
under examination. Screening was done using a High-Throughout
Screening (HTS) Luc assay with recombinant mycobacteria containing
a promoter fusion of a luciferase to the EB-inducible gene (Luc
assay). The Luc-assay and MIC assay are described in detail below.
These assays are well known to those skilled in the art. Based on
this initial screening, 300+ compound mixtures showed anti-TB
activity. FIG. 6 represents typical assay data in a luciferase
reporter strain containing an Rv0341 EMB-inducible promoter. FIG. 6
represents percent maximum Luminescense Count per Second (% Max.
LCPS) for pooled compound mixtures in one row (row D) in one of the
96-well plates.
Deconvolution of the Reactive Wells
The M. tuberculosis screening revealed approximately 300 active
compounds mixtures that were selected for deconvolution. In
particular, wells possessing activity of approximately <12.51M
in the HTS Luc assay, and/or an MIC of approximately <12.511M,
were selected for a total of 336 wells.
Deconvolutions were performed by discrete re-synthesis of each
substituted ethylene diamine compound in each active compound pool.
The pooled compounds in each active well were individually
synthesized, and screened. Syntheses of the targeted diamine
compounds in each active pool were done in the 96-well plates using
stored archived .alpha.-haloacetyl amides (resin attached
haloacetyl amides), according to the previously described reaction
steps (the addition of the second amine, the reduction with Red-A1,
and the cleavage from the solid support). The archived resins were
stored as individual compounds at 4.degree. C. The 96-well plates
were used for the remaining synthesis steps as previously
described.
The same screening tests, MIC and HTS Luc assay, were performed on
each deconvoluted compound. Representative Luminescence data for
deconvoluted compounds are shown in FIGS. 7 and 8. FIGS. 7 and 8
represent the Luminescence Count per Second (LCPS) for individual
compounds.
Summary of Screening Results
Overall, the deconvolution screening results revealed about 2,000
ethylene diamine compounds with inhibitory activity against M.
tuberculosis. More than 150 of these compounds exhibited MICs equal
to or lower than approximately 12.511M. FIG. 9 summarizes the MIC
data for all synthesized discrete compounds with an MIC of 50 .mu.M
or less. FIG. 10 summarizes Luc assay data for all compounds that
exhibit at least 10% activity at each concentration (the results
are not cumulative). The MIC and Luc activities were obtained for
non-purified samples, with chemical yields of approximately 20%,
based on an assumed 80% yield at each reaction step. In the Luc
assay, 32 compounds exhibited activity at 1.56 .mu.M, and in the
MIC assay, at least 11 compounds had an MIC of 3.13 .mu.M.
The total frequency of the top thirteen amines that contributed to
the activity of the substituted ethylene diamines are shown in FIG.
11, with each amine represented by its numerical designation. These
amines include the following: #11 2,3-Dimethylcylochexy amines #18
3,3-Diphenylpropylamine #44 1-Adamantanemethylamine #47
2,2-Diphenylethylamine #63 (S)-2-Amino-1-butanol #74.1
(-)-cis-Myrtanylamine #77.1 Cyclooctylamine #78.1 2-Adamantamine
#105a (1R,2R,3R,5S)-(-)-Isopinocampheylainine #231
2-Methoxyphenethylamine #255 (S)-Cylcohexylethylamine #266
Undecylamine #272 Geranylamine
Other amines that contributed to the activity of the substituted
ethylene diamines are shown in Table 2. The compounds in Table 2
are sorted by their MIC results. Some compounds, synthesized in
larger quantities (2-60 mg) on the Quests.RTM. Synthesizer, and
purified by HPLC using semi-preparative C18-column, are shown in
Table 3. Generally, the final purity of each compound in Table 3
was at least 90%.
TABLE-US-00002 TABLE 2 Synthetic Substituted Diethylene Diamines
Sorted by Minimum Inhibition Concentration MIC % N1 N2 R4 (uM)
Induction 3,3-Diphenylpropylamine exo-Aminonorbornane Hydrogen 3.13
53.70 2,2-Diphenylamine (+)-Isopinocampheylamine Hydrogen 3.13
93.94 2,2-Diphenylamine cis-(-)-Myrtanylamine Hydrogen 3.13 64.49
2,2-Diphenylamine Cyclooctylamine Hydrogen 3.13 63.44
2,2-Diphenylamine 3,4-Dihydroxynorephedrine Hydrogen 3.13 42.80
5-Aminoquinoline Cyclohexylamine Hydrogen 3.13 18.33
5-Aminoquinoline tert-Octylamine Hydrogen 3.13 20.85
5-Aminoquinoline 4-Methylcyclohexylamine Hydrogen 3.13 26.33
cis-(-)-Myrtanylamine (+)-Bornylamine Hydrogen 3.13 100.00
cis-(-)-Myrtanylamine 1-Adamantanemethylamine Hydrogen 3.13 85.20
cis-(-)-Myrtanylamine (-)-Isopinocampheylamine Hydrogen 3.13 60.94
1-Adamantanemethylamine tert-Octylamine Hydrogen 4.7 9.81
3,4-Dimethoxyphenethylamine 1-Adamantanemethylamine Hydrogen 6.25
11.45 3,4-Dimethoxyphenethylamine Hexetidine (mixture of isomers)
Hydrogen 6.25 0 3,4-Dimethoxyphenethylamine Dehydroabietylamine
Hydrogen 6.25 0 3,3-Diphenylpropylamine 1-Adamantanemethylamine
Hydrogen 6.25 9.53 3,3-Diphenylpropylamine 2-Methylcyclohexylamine
Hydrogen 6.25 50.08 (mix of cis and trans) 3,3-Diphenylpropylamine
1,3-Dimethylbutylamine Hydrogen 6.25 39.40 3,3-Diphenylpropylamine
1-(1-Adamantyl)ethylamine, Hydrogen 6.25 45.14 HCl
3,3-Diphenylpropylamine (S)-(-)-Cyclohexylethylamine Hydrogen 6.25
43.49 3,3-Diphenylpropylamine (R)-(-)-Cyclohexylethylamine Hydrogen
6.25 34.54 3,3-Diphenylpropylamine 1-Adamantanemethylamine Methyl
6.25 16.14 Propylamine Hexetidine (mixture of isomers) Hydrogen
6.25 0 Phenethylamine Hexetidine (mixture of isomers) Hydrogen 6.25
0 b-Methylphenethylamine Hexetidine (mixture of isomers) Hydrogen
6.25 0 b-Methylphenethylamine Undecylamine Hydrogen 6.25 0
2,2-Diphenylamine (+)-Bornylamine Hydrogen 6.25 87.86
2,2-Diphenylamine (-)-Isopinocampheylamine Hydrogen 6.25 77.80
2,2-Diphenylamine alpha-Methyltryptamine Hydrogen 6.25 55.07
2,2-Diphenylamine alpha-Methyltryptamine Hydrogen 6.25 23.08
2,2-Diphenylamine 4-Phenylbutylamine Hydrogen 6.25
2,2-Diphenylamine 2,5-Dimethoxyphenethylamine Hydrogen 6.25
2,2-Diphenylamine 2,4-Dichlorophenethylamine Hydrogen 6.25
2,2-Diphenylamine 2-(2-Aminomethyl) Hydrogen 6.25 phenylthio)benzyl
alcohol 2,2-Diphenylamine 1-(1-Naphthyl)ethylamine Hydrogen 6.25
7.20 Veratryl amine 2,5-Dimethoxyphenethylamine Hydrogen 6.25
Veratryl amine 2-(2-Aminomethyl) Hydrogen 6.25 phenylthio)benzyl
alcohol 5-Aminoquinoline 2-Aminoheptane Hydrogen 6.25 26.22
5-Aminoquinoline 1-Adamantanamine Hydrogen 6.25 18.91
1-Aminomethyl-1- Hexetidine (mixture of isomers) Hydrogen 6.25
cyclohexanol, HCl cis-(-)-Myrtanylamine 2,3-Dimethylcyclohexylamine
Hydrogen 6.25 100.00 cis-(-)-Myrtanylamine 3,3-Diphenylpropylamine
Hydrogen 6.25 87.78 cis-(-)-Myrtanylamine (+)-Isopinocampheylamine
Hydrogen 6.25 93.10 cis-(-)-Myrtanylamine 2,2-Diphenylamine
Hydrogen 6.25 81.84 cis-(-)-Myrtanylamine cis-(-)-Myrtanylamine
Hydrogen 6.25 68.24 cis-(-)-Myrtanylamine 1,3,3-Trimethyl-6-
Hydrogen 6.25 68.18 azabicyclo[3.2.1]octane cis-(-)-Myrtanylamine
1-Adamantanemethylamine Methyl 6.25 24.22 cis-(-)-Myrtanylamine
cis-(-)-Myrtanylamine Methyl 6.25 44.14 Cyclooctylamine
3,3-Diphenylpropylamine Hydrogen 6.25 100.00 Cyclooctylamine
(-)-Isopinocampheylamine Hydrogen 6.25 59.13 sec-Butylamine
Hexetidine (mixture of isomers) Hydrogen 6.25 3-Methylbenzylamine
Hexetidine (mixture of isomers) Hydrogen 6.25 3-Methylbenzylamine
Undecylamine Hydrogen 6.25 2-Methoxyethylamine Hexetidine (mixture
of isomers) Hydrogen 6.25 Geranylamine 2-Adamantanamine, HCl
Hydrogen 6.25 25.66 1-Adamantanemethylamine 4-Benzylpiperidine
Hydrogen 9.4 0 1-Adamantanemethylamine 2,3-Dimethylcyclohexylamine
Hydrogen 9.4 0 1-Adamantanemethylamine 3,3-Diphenylpropylamine
Hydrogen 9.4 40.06 1-Adamantanemethylamine 1-Adamantanemethylamine
Hydrogen 9.4 15.25 1-Adamantanemethylamine 2,2-Diphenylamine
Hydrogen 9.4 0 1-Adamantanemethylamine 1,3,3-Trimethyl-6- Hydrogen
9.4 0 azabicyclo[3.2.1]octane 1-Adamantanemethylamine 138 Hydrogen
9.4 0 3-Phenyl-1-propylamine 138 Hydrogen 9.4 2,2-Diphenylamine
1-Adamantanemethylamine Hydrogen 9.4 65.89 2,2-Diphenylamine 138
Hydrogen 9.4 Furfurylamine Hexetidine (mixture of isomers) Hydrogen
12.5 0 3,4,5-Trimethoxybenzylamine Hexetidine (mixture of isomers)
Hydrogen 12.5 0 1-Methyl-3-phenylpropylamine Dehydroabietylamine
Hydrogen 12.5 0 Cyclobutylamine Hexetidine (mixture of isomers)
Hydrogen 12.5 0 2-Fluorobenzylamine Hexetidine (mixture of isomers)
Hydrogen 12.5 0 2-Fluorobenzylamine Dehydroabietylamine Hydrogen
12.5 0 3,4-Dimethoxyphenethylamine Undecylamine Hydrogen 12.5 0
3,3-Diphenylpropylamine exo-Aminonorbornane Hydrogen 12.5 14.38
3,3-Diphenylpropylamine Decahydroquinoline Hydrogen 12.5 22.52
3,3-Diphenylpropylamine Hexetidine (mixture of isomers) Hydrogen
12.5 0 3,3-Diphenylpropylamine 4-Phenylbutylamine Hydrogen 12.5 0
3,3-Diphenylpropylamine 2-Methoxyphenethylamine Hydrogen 12.5 6.82
3,3-Diphenylpropylamine 2,4-Dichlorophenethylamine Hydrogen 12.5 0
3,3-Diphenylpropylamine 1-Aminoindan Hydrogen 12.5 18.05
3,3-Diphenylpropylamine Undecylamine Hydrogen 12.5 0
3,3-Diphenylpropylamine Dehydroabietylamine Hydrogen 12.5 0
3,3-Diphenylpropylamine 2-(1-Cyclohexenyl)ethylamine Methyl 12.5
9.5 3,3-Diphenylpropylamine cis-(-)-Myrtanylamine Methyl 12.5 18.41
3,3-Diphenylpropylamine Cyclooctylamine Methyl 12.5 20.84
Propylamine Dehydroabietylamine Hydrogen 12.5 0 Phenethylamine
Dehydroabietylamine Hydrogen 12.5 0 Cyclohexylamine Hexetidine
(mixture of isomers) Hydrogen 12.5 0 3-Amino-1-propanol Hexetidine
(mixture of isomers) Hydrogen 12.5 0 b-Methylphenethylamine
Dehydroabietylamine Hydrogen 12.5 0 4-Methoxyphenethylamine
2-Fluorophenethylamine Hydrogen 12.5 0 4-Methoxyphenethylamine
2-(1-Cyclohexenyl)ethylamine Hydrogen 12.5 0
4-Methoxyphenethylamine 2,4-Dimethoxybenzylamine Hydrogen 12.5 0
4-Methoxyphenethylamine 4-Fluorophenethylamine Hydrogen 12.5 16.78
4-Methoxyphenethylamine Hexetidine (mixture of isomers) Hydrogen
12.5 0 Tetrahydrofurfurylamine Hexetidine (mixture of isomers)
Hydrogen 12.5 0 Amylamine 4-Fluorophenethylamine Hydrogen 12.5 0
3-Phenyl-1-propylamine 2-(1-Cyclohexenyl)ethylamine Hydrogen 12.5
3-Phenyl-1-propylamine 4-Fluorophenethylamine Hydrogen 12.5 12.94
2,2-Diphenylamine tert-Amylamine Hydrogen 12.5 9.05
2,2-Diphenylamine Undecylamine Hydrogen 12.5 2,2-Diphenylamine
Dehydroabietylamine Hydrogen 12.5 2,2-Diphenylamine
cis-(-)-Myrtanylamine Methyl 12.5 45.18 1-(3-Aminopropyl)-2-
2,5-Dimethoxyphenethylamine Hydrogen 12.5 pyrrolidinone (tech)
1-(3-Aminopropyl)-2- 2-(2- Hydrogen 12.5 pyrrolidinone (tech)
Aminomethyl)phenylthio)benzyl alcohol
4-(Trifluoromethyl)benzylamine 2,5-Dimethoxyphenethylamine Hydrogen
12.5 4-(Trifluoromethyl)benzylamine 1-(1-Naphthyl)ethylamine
Hydrogen 12.5 Veratryl amine 4-Phenylbutylamine Hydrogen 12.5
5-Amino-1-pentanol 2,5-Dimethoxyphenethylamine Hydrogen 12.5
5-Amino-1-pentanol 2-(2- Hydrogen 12.5
Aminomethyl)phenylthio)benzyl alcohol 2-(1-Cyclohexenyl)ethylamine
2-(1-Cyclohexenyl)ethylamine Hydrogen 12.5
2-(1-Cyclohexenyl)ethylamine 4-Fluorophenethylamine Hydrogen 12.5
2-(1-Cyclohexenyl)ethylamine 4-Phenylbutylamine Hydrogen 12.5
2-(1-Cyclohexenyl)ethylamine 2,5-Dimethoxyphenethylamine Hydrogen
12.5 2-(1-Cyclohexenyl)ethylamine 2-(2-Aminomethyl) Hydrogen 12.5
phenylthio)benzyl alcohol 1-Aminomethyl-1-
2,5-Dimethoxyphenethylamine Hydrogen 12.5 cyclohexanol, HCl
3-Fluorobenzylamine 2,5-Dimethoxyphenethylamine Hydrogen 12.5
4-Amino-1-butanol Hexetidine (mixture of isomers) Hydrogen 12.5
2-Ethoxybenzylamine Hexetidine (mixture of isomers) Hydrogen 12.5
cis-(-)-Myrtanylamine Cyclooctylamine Hydrogen 12.5 67.73
cis-(-)-Myrtanylamine 4-Methylcyclohexylamine Hydrogen 12.5 18.39
cis-(-)-Myrtanylamine 1-Adamantanamine Hydrogen 12.5 60.16
cis-(-)-Myrtanylamine 3,3-Diphenylpropylamine Methyl 12.5 22.32
Cyclooctylamine (+)-Isopinocampheylamine Hydrogen 12.5 57.83
Cyclooctylamine (+)-Bornylamine Hydrogen 12.51 100.00
Cyclooctylamine 1-Adamantanemethylamine Hydrogen 12.5 52.95
Cyclooctylamine 2,2-Diphenylamine Hydrogen 12.5 71.43
Cyclooctylamine cis-(-)-Myrtanylamine Hydrogen 12.5 84.56
Cyclooctylamine Cyclooctylamine Hydrogen 12.5 59.21 Cyclooctylamine
Hexetidine (mixture of isomers) Hydrogen 12.5 Cyclooctylamine
Aminodiphenylmethane Hydrogen 12.5 Cyclooctylamine Undecylamine
Hydrogen 12.5 5.61 Cyclooctylamine 3,3-Diphenylpropylamine Methyl
12.5 53.92 Cyclooctylamine (+)-Isopinocampheylamine Methyl 12.5
Cyclooctylamine cis-(-)-Myrtanylamine Methyl 12.5 33.89
4-Chlorophenylalaninol Hexetidine (mixture of isomers) Hydrogen
12.5 (-)-Isopinocampheylamine 3,3-Diphenylpropylamine Hydrogen 12.5
23.68 (-)-Isopinocampheylamine (+)-Bornylamine Hydrogen 12.5 44.85
(-)-Isopinocampheylamine 2-Amino-1-propanol, d,1 Hydrogen 12.5
46.19 (-)-Isopinocampheylamine cis-(-)-Myrtanylamine Hydrogen 12.5
33.87 (-)-Isopinocampheylamine 2-Adamantanamine, HCl Hydrogen 12.5
24.29 (-)-Isopinocampheylamine Aminodiphenylmethane Hydrogen 12.5
48.35 Allylamine Hexetidine (mixture of isomers) Hydrogen 12.5
3-Ethoxypropylamine Hexetidine (mixture of isomers) Hydrogen 12.5
sec-Butylamine Dehydroabietylamine Hydrogen 12.5 2-Aminoheptane
Dehydroabietylamine Hydrogen 12.5 Ethanolamine Hexetidine (mixture
of isomers) Hydrogen 12.5 3-Methylbenzylamine 4-Phenylbutylamine
Hydrogen 12.5 3-Methylbenzylamine 2,4-Dichlorophenethylamine
Hydrogen 12.5 3-Methylbenzylamine Dehydroabietylamine Hydrogen 12.5
Piperonylamine Hexetidine (mixture of isomers) Hydrogen 12.5
Piperonylamine Dehydroabietylamine Hydrogen 12.5
2-Methoxyethylamine Dehydroabietylamine Hydrogen 12.5
4-Fluorophenethylamine Hexetidine (mixture of isomers) Hydrogen
12.5 3-o-Methyldopamine, HCl Hexetidine (mixture of isomers)
Hydrogen 12.5 3-o-Methyldopamine, HCl Undecylamine Hydrogen 12.5
3-o-Methyldopamine, HCl Dehydroabietylamine Hydrogen 12.5
3-Fluorophenethylamine Hexetidine (mixture of isomers) Hydrogen
12.5 3-Fluorophenethylamine Dehydroabietylamine Hydrogen 12.5
2-Methoxyphenethylamine Hexetidine (mixture of isomers) Hydrogen
12.5 2-Methoxyphenethylamine Aminodiphenylmethane Hydrogen 12.5
34.67 2-Fluoroethylamine, HCl Hexetidine (mixture of isomers)
Hydrogen 12.5 2-Amino-1-phenylethanol Hexetidine (mixture of
isomers) Hydrogen 12.5 2-Amino-1-phenylethanol Dehydroabietylamine
Hydrogen 12.5 2,5-Dimethoxyphenethylamine 2-Adamantanamine, HCl
Hydrogen 12.5 22.18 2-(2-Chlorophenyl)ethylamine
N-Allylcyclopentylamine Hydrogen 12.5 62.31
2-(2-Chlorophenyl)ethylamine Hexetidine (mixture of isomers)
Hydrogen 12.5 3-Hydroxytyramine Hexetidine (mixture of isomers)
Hydrogen 12.5 4- 2-Adamantanamine, HCl Hydrogen 12.5 28.34
(Trifluoromethoxy)benzylamine Geranylamine (+)-Bornylamine Hydrogen
12.5 Geranylamine 1,3,3-Trimethyl-6- Hydrogen 12.5 37.42
azabicyclo[3.2.1]octane Geranylamine 2-Ethylpiperidine Hydrogen
12.5 29.81 Geranylamine 1-Adamantanamine Hydrogen 12.5 16.63
Geranylamine N-Allylcyclopentylamine Hydrogen 12.5 74.86
Geranylamine Aminodiphenylmethane Hydrogen 12.5 57.93 Geranylamine
Dehydroabietylamine Hydrogen 12.5 1-Adamantanemethylamine
Decahydroquinoline Hydrogen 18.8 0 1-Adamantanemethylamine
1-Adamantanamine Hydrogen 18.8 0 2,2-Diphenylamine
2,3-Dimethylcyclohexylamine Hydrogen 18.8 23.60 2,2-Diphenylamine
tert-Octylamine Hydrogen 18.8 19.29 2,2-Diphenylamine
Decahydroquinoline Hydrogen 18.8 8.96 4-Methylbenzylamine
Furfurylamine Hydrogen 25 13.46 4-Methylbenzylamine Benzylamine
Hydrogen 25 17.07 4-Methylbenzylamine Hexetidine (mixture of
isomers) Hydrogen 25 0 4-Methylbenzylamine Dehydroabietylamine
Hydrogen 25 0 Cyclopentylamine Hexetidine (mixture of isomers)
Hydrogen 25 0 Cyclopentylamine Dehydroabietylamine Hydrogen 25 0
Furfurylamine Furfurylamine Hydrogen 25 0
1-Methyl-3-phenylpropylamine Hexetidine (mixture of isomers)
Hydrogen 25 0 1-Methyl-3-phenylpropylamine Undecylamine Hydrogen 25
0 1,2,3,4-Tetrahydro-1- Undecylamine Hydrogen 25 6.24 naphthylamine
1,2,3,4-Tetrahydro-1- Dehydroabietylamine Hydrogen 25 0
naphthylamine 2,3-Dimethylcyclohexylamine Undecylamine Hydrogen 25
0 2,3-Dimethylcyclohexylamine Dehydroabietylamine Hydrogen 25 0
Tyramine Hexetidine (mixture of isomers) Hydrogen 25 0 Tyramine
Undecylamine Hydrogen 25 0 Tyramine Dehydroabietylamine Hydrogen 25
0 Tyramine cis-(-)-Myrtanylamine Methyl 25 0 2-Fluorobenzylamine
Undecylamine Hydrogen 25 0 (R)-2-Amino-1-butanol Hexetidine
(mixture of isomers) Hydrogen 25 0 3,3-Diphenylpropylamine
(S)-(+)-1-Amino-2-propanol Hydrogen 25 0 3,3-Diphenylpropylamine
2-Ethylpiperidine Hydrogen 25 11.32 3,3-Diphenylpropylamine
N-Allylcyclopentylamine Hydrogen 25 11.63 3,3-Diphenylpropylamine
Aminodiphenylmethane Hydrogen 25 0 3,3-Diphenylpropylamine
3,5-Dimethylpiperidine (cis- Hydrogen 25 30.28 and trans-)
3,3-Diphenylpropylamine Allylcyclohexylamine Hydrogen 25 9.10
Propylamine Undecylamine Hydrogen 25 0 Phenethylamine Undecylamine
Hydrogen 25 0 Tryptamine (S)-(+)-1-Amino-2-propanol Hydrogen 25 0
Tryptamine 2-Amino-2-methyl-1-propanol Hydrogen 25 0
Cyclohexylamine Undecylamine Hydrogen 25 0 Cyclohexylamine
Dehydroabietylamine Hydrogen 25 0 (+)-Isopinocampheylamine
Dehydroabietylamine Hydrogen 25 0 Benzylamine Hexetidine (mixture
of isomers) Hydrogen 25 Benzylamine Undecylamine Hydrogen 25
3-Amino-1-propanol Dehydroabietylamine Hydrogen 25 0
2-Fluorophenethylamine 2-Fluorophenethylamine Hydrogen 25 0
2-Fluorophenethylamine Veratryl amine Hydrogen 25 0
2-Fluorophenethylamine 2,4-Dimethoxybenzylamine Hydrogen 25 0
2-Fluorophenethylamine 2-Amino-2-methyl-1-propanol Hydrogen 25 0
2-Fluorophenethylamine 4-Fluorophenethylamine Hydrogen 25 0
2-Fluorophenethylamine Hexetidine (mixture of isomers) Hydrogen 25
0 2-Fluorophenethylamine 1-(1-Naphthyl)ethylamine Hydrogen 25 0
2-Fluorophenethylamine 1-Adamantanemethylamine Methyl 25 3.21
2-Fluorophenethylamine cis-(-)-Myrtanylamine Methyl 25 4.89
b-Methylphenethylamine 4-Phenylbutylamine Hydrogen 25 0
b-Methylphenethylamine 2,4-Dichlorophenethylamine Hydrogen 25 0
b-Methylphenethylamine 1-(1-Naphthyl)ethylamine Hydrogen 25 0
4-Methoxyphenethylamine 1-Adamantanemethylamine Hydrogen 25 0
4-Methoxyphenethylamine 1-(3-Aminopropyl)-2- Hydrogen 25 0
pyrrolidinone (tech) 4-Methoxyphenethylamine Veratryl amine
Hydrogen 25 0 4-Methoxyphenethylamine Undecylamine Hydrogen 25 0
4-Methoxyphenethylamine Dehydroabietylamine Hydrogen 25 0
Tetrahydrofurfurylamine Dehydroabietylamine Hydrogen 25 0 Amylamine
2-Fluorophenethylamine Hydrogen 25 0 Amylamine
2-(1-Cyclohexenyl)ethylamine Hydrogen 25 0 Amylamine
2,4-Dimethoxybenzylamine Hydrogen 25 0 3-Phenyl-1-propylamine
2-Fluorophenethylamine Hydrogen 25 3-Phenyl-1-propylamine
1-Adamantanemethylamine Hydrogen 25 3-Phenyl-1-propylamine
2,4-Dimethoxybenzylamine Hydrogen 25 3-Phenyl-1-propylamine
Hexetidine (mixture of isomers) Hydrogen 25 3-Phenyl-1-propylamine
4-Phenylbutylamine Hydrogen 25 3-Phenyl-1-propylamine
2,4-Dichlorophenethylamine Hydrogen 25 3-Phenyl-1-propylamine
Undecylamine Hydrogen 25 3-Phenyl-1-propylamine Dehydroabietylamine
Hydrogen 25 2,2-Diphenylamine 4-(2-Aminoethyl)morpholine Hydrogen
25 2,2-Diphenylamine 1-(3-Aminopropyl)-2- Hydrogen 25 pyrrolidinone
(tech) 2,2-Diphenylamine 2-(1-Cyclohexenyl)ethylamine Hydrogen 25
2,2-Diphenylamine 2,4-Dimethoxybenzylamine Hydrogen 25
2,2-Diphenylamine 4-(3-Aminopropyl)morpholine Hydrogen 25
2,2-Diphenylamine 4-Fluorophenethylamine Hydrogen 25
2,2-Diphenylamine Hexetidine (mixture of isomers) Hydrogen 25
2,2-Diphenylamine (S)-(-)-Cyclohexylethylamine Hydrogen 25
2,2-Diphenylamine 1-Adamantanemethylamine Methyl 25 5.84
1-(3-Aminopropyl)-2- 4-Phenylbutylamine Hydrogen 25 pyrrolidinone
(tech) 4-(Trifluoromethyl)benzylamine 1-Adamantanemethylamine
Hydrogen 25 4-(Trifluoromethyl)benzylamine tert-Amylamine Hydrogen
25 4-(Trifluoromethyl)benzylamine alpha-Methyltryptamine Hydrogen
25 6.06 4-(Trifluoromethyl)benzylamine 4-Phenylbutylamine Hydrogen
25 4-(Trifluoromethyl)benzylamine 2-(2-Aminomethyl) Hydrogen 25
5.13 phenylthio)benzyl alcohol 4-(Trifluoromethyl)benzylamine
Undecylamine Hydrogen 25 4-(Trifluoromethyl)benzylamine
(-)-3,4-Dihydroxynorephedrine Hydrogen 25
4-(Trifluoromethyl)benzylamine Dehydroabietylamine Hydrogen 25
Veratryl amine tert-Amylamine Hydrogen 25 5-Amino-1-pentanol
4-Phenylbutylamine Hydrogen 25 2-(1-Cyclohexenyl)ethylamine
2-Fluorophenethylamine Hydrogen 25 2-(1-Cyclohexenyl)ethylamine
1-Adamantanemethylamine Hydrogen 25 1-Aminomethyl-1-
4-Phenylbutylamine Hydrogen 25 cyclohexanol, HCl
3-Fluorobenzylamine 4-Phenylbutylamine Hydrogen 25
3-Fluorobenzylamine 2-(2- Hydrogen 25 Aminomethyl)phenylthio)benzyl
alcohol 2,4-Dimethoxybenzylamine 1-Adamantanamine Hydrogen 25
2,4-Dimethoxybenzylamine Hexetidine (mixture of isomers) Hydrogen
25 2,4-Dimethoxybenzylamine Undecylamine Hydrogen 25
2,4-Dimethoxybenzylamine Dehydroabietylamine Hydrogen 25
2-Ethoxybenzylamine 1-Adamantanamine Hydrogen 25
2-Ethoxybenzylamine N-Phenylethyldiamine Hydrogen 25
2-Ethoxybenzylamine 2,4-Dichlorophenethylamine Hydrogen 25
2-Ethoxybenzylamine 2-(2-Chlorophenyl)ethylamine Hydrogen 25 3.89
2-Ethoxybenzylamine Undecylamine Hydrogen 25 2-Ethoxybenzylamine
Dehydroabietylamine Hydrogen 25 cis-(-)-Myrtanylamine
2-(1-Cyclohexenyl)ethylamine Hydrogen 25 cis-(-)-Myrtanylamine
Hexetidine (mixture of isomers) Hydrogen 25 cis-(-)-Myrtanylamine
Aminodiphenylmethane Hydrogen 25 cis-(-)-Myrtanylamine
2,4-Dichlorophenethylamine Hydrogen 25 cis-(-)-Myrtanylamine
(S)-(-)-Cyclohexylethylamine Hydrogen 25 28.94
cis-(-)-Myrtanylamine Undecylamine Hydrogen 25
cis-(-)-Myrtanylamine (+)-Isopinocampheylamine Methyl 25
cis-(-)-Myrtanylamine Cyclooctylamine Methyl 25 24.92
Cyclooctylamine 2,3-Dimethylcyclohexylamine Hydrogen 25 50.55
Cyclooctylamine (S)-2-Amino-1-butanol Hydrogen 25 100.00
Cyclooctylamine 2-Adamantanamine, HCl Hydrogen 25 29.61
Cyclooctylamine 4-Phenylbutylamine Hydrogen 25 Cyclooctylamine
2-Chlorobenzylamine Hydrogen 25 Cyclooctylamine 2-Aminoindan, HCl
Hydrogen 25 Cyclooctylamine Dehydroabietylamine Hydrogen 25
Cyclooctylamine 1-(1-Naphthyl)ethylamine Hydrogen 25 4.62
Cyclooctylamine 1-Adamantanemethylamine Methyl 25 14.20
2,3-Dimethoxybenzylamine Hexetidine (mixture of isomers) Hydrogen
25 2,3-Dimethoxybenzylamine Undecylamine Hydrogen 25
2,3-Dimethoxybenzylamine Dehydroabietylamine Hydrogen 25
4-Methylcyclohexylamine Hexetidine (mixture of isomers) Hydrogen 25
4-Methylcyclohexylamine Undecylamine Hydrogen 25
4-Methylcyclohexylamine Dehydroabietylamine Hydrogen 25
4-Fluorobenzylamine Dibenzylamine Hydrogen 25 27.98 trans-2-
Cyclooctylamine Hydrogen 25 32.80 Phenylcyclopropylamine, HCl
trans-2- 2-Adamantanamine, HCl Hydrogen 25 18.99
Phenylcyclopropylamine, HCl trans-2- 1-Adamantanamine Hydrogen 25
18.84 Phenylcyclopropylamine, HCl Thiomicamine Hexetidine (mixture
of isomers) Hydrogen 25 (R)-1-Amino-2-propanol Hexetidine (mixture
of isomers) Hydrogen 25 4-Chlorophenylalaninol
2,4-Dichlorophenethylamine Hydrogen 25 4-Chlorophenylalaninol
Undecylamine Hydrogen 25 4-Chlorophenylalaninol Dehydroabietylamine
Hydrogen 25 I-Leucinol Hexetidine (mixture of isomers) Hydrogen 25
I-Leucinol 2,4-Dichlorophenethylamine Hydrogen 25 I-Leucinol
Dehydroabietylamine Hydrogen 25 (-)-Isopinocampheylamine
2-Methoxyphenethylamine Hydrogen 25 29.59 (-)-Isopinocampheylamine
Undecylamine Hydrogen 25 Allylamine Dehydroabietylamine Hydrogen 25
3-Amino-1,2-propanediol Hexetidine (mixture of isomers) Hydrogen 25
3-Ethoxypropylamine 3,3-Diphenylpropylamine Hydrogen 25
3-Ethoxypropylamine Undecylamine Hydrogen 25 3-Ethoxypropylamine
Dehydroabietylamine Hydrogen 25 sec-Butylamine
2,4-Dichlorophenethylamine Hydrogen 25 sec-Butylamine Undecylamine
Hydrogen 25 2-Aminoheptane Hexetidine (mixture of isomers) Hydrogen
25 2-Aminoheptane 4-Phenylbutylamine Hydrogen 25 2-Aminoheptane
2,4-Dichlorophenethylamine Hydrogen 25 1-Naphthalenemethylamine
Hexetidine (mixture of isomers) Hydrogen 25
1-Naphthalenemethylamine 4-Phenylbutylamine Hydrogen 25
1-Naphthalenemethylamine 2,4-Dichlorophenethylamine Hydrogen 25
1-Naphthalenemethylamine Undecylamine Hydrogen 25 Ethanolamine
Dehydroabietylamine Hydrogen 25 Piperonylamine 4-Phenylbutylamine
Hydrogen 25 1-Ethylpropylamine Hexetidine (mixture of isomers)
Hydrogen 25 1-Ethylpropylamine Dehydroabietylamine Hydrogen 25
Isopropylamine Hexetidine (mixture of isomers) Hydrogen 25
4-Fluorophenethylamine 4-Phenylbutylamine Hydrogen 25
4-Fluorophenethylamine 2,4-Dichlorophenethylamine Hydrogen 25
4-Fluorophenethylamine Dehydroabietylamine Hydrogen 25
3-Fluorophenethylamine Undecylamine Hydrogen 25
2-Thiopheneethylamine 2-Adamantanamine, HCl Hydrogen 25 19.09
2-Methylcyclohexylamine (mix Hexetidine (mixture of isomers)
Hydrogen 25 of cis and trans) 2-Methylcyclohexylamine (mix
Dehydroabietylamine Hydrogen 25 of cis and trans)
2-Methoxyphenethylamine 2-Adamantanamine, HCl Hydrogen 25 26.77
2-Methoxyphenethylamine (-)-Isopinocampheylamine Hydrogen 25 31.95
2-Methoxyphenethylamine 1-Adamantanamine Hydrogen 25 24.38
2-Methoxyphenethylamine N-Allylcyclopentylamine Hydrogen 25 14.56
2-Methoxyphenethylamine 4-Phenylbutylamine Hydrogen 25
2-Methoxyphenethylamine Undecylamine Hydrogen 25
2-Methoxyphenethylamine Dehydroabietylamine Hydrogen 25
2-Fluoroethylamine, HCl Undecylamine Hydrogen 25
2-Fluoroethylamine, HCl Dehydroabietylamine Hydrogen 25
2-Aminoindan, HCl 2-Adamantanamine, HCl Hydrogen 25 17.72
2-Amino-1-phenylethanol Undecylamine Hydrogen 25
2,5-Dimethoxyphenethylamine (+)-Bornylamine Hydrogen 25 25.78
2,5-Dimethoxyphenethylamine Noradamantamine, HCl Hydrogen 25 11.73
2,5-Dimethoxyphenethylamine 1-Adamantanamine Hydrogen 25 12.57
2-(2-Chlorophenyl)ethylamine 4-Phenylbutylamine Hydrogen 25
2-(2-Chlorophenyl)ethylamine Undecylamine Hydrogen 25
2-(2-Chlorophenyl)ethylamine 1-(1-Naphthyl)ethylamine Hydrogen 25
2-(2- Hexetidine (mixture of isomers) Hydrogen 25
Aminomethyl)phenylthio)benzyl alcohol 2-(2- 4-Phenylbutylamine
Hydrogen 25 Aminomethyl)phenylthio)benzyl alcohol 2-(2-
Undecylamine Hydrogen 25 Aminomethyl)phenylthio)benzyl alcohol
1-Aminoindan Hexetidine (mixture of isomers) Hydrogen 25
1-Aminoindan Undecylamine Hydrogen 25 1-Aminoindan
Dehydroabietylamine Hydrogen 25 1,3-Dimethylbutylamine Hexetidine
(mixture of isomers) Hydrogen 25 1,3-Dimethylbutylamine
Undecylamine Hydrogen 25 5.92 1,3-Dimethylbutylamine
Dehydroabietylamine Hydrogen 25 (S)-(-)-Cyclohexylethylamine
(-)-Isopinocampheylamine Hydrogen 25 19.31
(S)-(-)-Cyclohexylethylamine Hexetidine (mixture of isomers)
Hydrogen 25 (S)-(-)-Cyclohexylethylamine Undecylamine Hydrogen 25
10.88 (S)-(-)-Cyclohexylethylamine Dehydroabietylamine Hydrogen 25
(S)-(-)-2-Amino-3-phenyl-1- Hexetidine (mixture of isomers)
Hydrogen 25 propanol (S)-(-)-2-Amino-3-phenyl-1- Undecylamine
Hydrogen 25 propanol (S)-(-)-2-Amino-3-phenyl-1-
Dehydroabietylamine Hydrogen 25 propanol (1S,2S)-(+)-2-Amino-3-
Hexetidine (mixture of isomers) Hydrogen 25
methoxy-1-phenyl-1-propanol Octadecylamine (+)-Bornylamine Hydrogen
25 Octadecylamine 1-Adamantanamine Hydrogen 25 Geranylamine
2,3-Dimethylcyclohexylamine Hydrogen 25 14.53 Geranylamine
tert-Octylamine Hydrogen 25 15.22 Geranylamine
1-Adamantanemethylamine Hydrogen 25 4.37 Geranylamine
Decahydroquinoline Hydrogen 25 31.79 Geranylamine Dibenzylamine
Hydrogen 25 6.48 Geranylamine N-Butylbenzylamine Hydrogen 25 16.44
Geranylamine Cyclooctylamine Hydrogen 25 12.37 Geranylamine
(-)-Isopinocampheylamine Hydrogen 25 8.95 Geranylamine
1-(1-Adamantyl)ethylamine, Hydrogen 25 32.95 HCl Geranylamine
Undecylamine Hydrogen 25 Geranylamine 1-(1-Naphthyl)ethylamine
Hydrogen 25 Amylamine 1-Adamantanamine Hydrogen 37.5 0
3-Phenyl-1-propylamine 3,3-Diphenylpropylamine Hydrogen 37.5
3-Phenyl-1-propylamine 2,2-Diphenylamine Hydrogen 37.5
3-Phenyl-1-propylamine 1-Adamantanamine Hydrogen 37.5 18.65
2,2-Diphenylamine 3,3-Diphenylpropylamine Hydrogen 37.5
2,2-Diphenylamine 2,2-Diphenylamine Hydrogen 37.5 5.56
2,2-Diphenylamine 1,3,3-Trimethyl-6- Hydrogen 37.5 8.67
azabicyclo[3.2.1]octane 2,2-Diphenylamine 1-Adamantanamine Hydrogen
37.5 58.10 4-(Trifluoromethyl)benzylamine tert-Octylamine Hydrogen
37.5 7.47 4-(Trifluoromethyl)benzylamine 138 Hydrogen 37.5
4-Methylbenzylamine 2-Fluorobenzylamine Hydrogen 50 22.10
4-Methylbenzylamine 4-Fluorobenzylamine Hydrogen 50 14.62
4-Methylbenzylamine alpha-Methyltryptamine Hydrogen 50 0
4-Methylbenzylamine Undecylamine Hydrogen 50 0 Cyclopentylamine
Undecylamine Hydrogen 50 0 Furfurylamine 2-Fluorobenzylamine
Hydrogen 50 0 Furfurylamine Benzylamine Hydrogen 50 0 Furfurylamine
4-Fluorobenzylamine Hydrogen 50 0 Furfurylamine
alpha-Methyltryptamine Hydrogen 50 0 Furfurylamine Undecylamine
Hydrogen 50 0 Furfurylamine Dehydroabietylamine Hydrogen 50 0
Furfurylamine Furfurylamine Hydrogen 50 0
3,4,5-Trimethoxybenzylamine 2-Fluorobenzylamine Hydrogen 50 0
3,4,5-Trimethoxybenzylamine Benzylamine Hydrogen 50 0
3,4,5-Trimethoxybenzylamine alpha-Methyltryptamine Hydrogen 50 0
3,4,5-Trimethoxybenzylamine Undecylamine Hydrogen 50 0
3,4,5-Trimethoxybenzylamine Dehydroabietylamine Hydrogen 50 0
1-Methyl-3-phenylpropylamine alpha-Methyltryptamine Hydrogen 50 0
1-Methyl-3-phenylpropylamine Octadecylamine Hydrogen 50 0
Cyclobutylamine Octadecylamine Hydrogen 50 0 Cyclobutylamine
Undecylamine Hydrogen 50 0 Cyclobutylamine Dehydroabietylamine
Hydrogen 50 0 1,2,3,4-Tetrahydro-1- Hexetidine (mixture of isomers)
Hydrogen 50 0 naphthylamine 1,2,3,4-Tetrahydro-1-
Aminodiphenylmethane Hydrogen 50 4.31 naphthylamine
1,2,3,4-Tetrahydro-1- alpha-Methyltryptamine Hydrogen 50 0
naphthylamine 1,2,3,4-Tetrahydro-1- 2-Methoxyphenethylamine
Hydrogen 50 0 naphthylamine 2,3-Dimethylcyclohexylamine Hexetidine
(mixture of isomers) Hydrogen 50 0 2,3-Dimethylcyclohexylamine
Aminodiphenylmethane Hydrogen 50 3.64 2,3-Dimethylcyclohexylamine
alpha-Methyltryptamine Hydrogen 50 0 Tyramine Furfurylamine
Hydrogen 50 0 Tyramine 2-Fluorobenzylamine Hydrogen 50 4.07
Tyramine Benzylamine Hydrogen 50 0 Tyramine
2,4-Dichlorophenethylamine Hydrogen 50 0 2-Fluorobenzylamine
Aminodiphenylmethane Hydrogen 50 0 2-Fluorobenzylamine
4-Phenylbutylamine Hydrogen 50 0 2-Fluorobenzylamine
2-Methoxyphenethylamine Hydrogen 50 0 2-Fluorobenzylamine
2,4-Dichlorophenethylamine Hydrogen 50 0 2-Fluorobenzylamine
1,3-Dimethylbutylamine Hydrogen 50 0 2-Fluorobenzylamine
1-(1-Adamantyl)ethylamine, Hydrogen 50 0 HCl (R)-2-Amino-1-butanol
Dehydroabietylamine Hydrogen 50 0 3,4-Dimethoxyphenethylamine
Aminodiphenylmethane Hydrogen 50 0 3,4-Dimethoxyphenethylamine
4-Phenylbutylamine Hydrogen 50 0 3,4-Dimethoxyphenethylamine
2-Methoxyphenethylamine Hydrogen 50 0
3,4-Dimethoxyphenethylamine 2,4-Dichlorophenethylamine Hydrogen 50
0 3,4-Dimethoxyphenethylamine 1,3-Dimethylbutylamine Hydrogen 50 0
3,3-Diphenylpropylamine Piperidine Hydrogen 50 0
3,3-Diphenylpropylamine 2,3-Dimethylcyclohexylamine Methyl 50 7.81
3,3-Diphenylpropylamine (-)-Isopinocamphenylamine Methyl 50 13.06
Propylamine (S)-(+)-1-Amino-2-propanol Hydrogen 50 0 Phenethylamine
(S)-(+)-1-Amino-2-propanol Hydrogen 50 0 Phenethylamine
4-Phenylbutylamine Hydrogen 50 0 Phenethylamine
2,4-Dichlorophenethylamine Hydrogen 50 0 Phenethylamine
1,3-Dimethylbutylamine Hydrogen 50 0 Phenethylamine
1-(1-Adamantyl)ethylamine, Hydrogen 50 0 HCl Phenethylamine
1-(1-Naphthyl)ethylamine Hydrogen 50 0 4-(2-Aminoethyl)morpholine
2-Amino-2-methyl-1-propanol Hydrogen 50 0 Cyclohexylamine
2,4-Dichlorophenethylamine Hydrogen 50 0 exo-Aminonorbornane
Benzylamine Hydrogen 50 0 (+)-Isopinocampheylamine Hexetidine
(mixture of isomers) Hydrogen 50 0 (+)-Isopinocampheylamine
Aminodiphenylmethane Hydrogen 50 5.07 (+)-Isopinocampheylamine
4-Phenylbutylamine Hydrogen 50 0 (+)-Isopinocampheylamine
2,4-Dichlorophenethylamine Hydrogen 50 0 (+)-Isopinocampheylamine
Undecylamine Hydrogen 50 0 Benzylamine 3,3-Diphenylpropylamine
Hydrogen 50 Benzylamine 2-Amino-2-methyl-1-propanol Hydrogen 50
Benzylamine 1-(1-Naphthyl)ethylamine Hydrogen 50 Benzylamine
2,4-Dichlorophenethylamine Hydrogen 50 3-Amino-1-propanol
Undecylamine Hydrogen 50 0 2-Fluorophenethylamine
3,3-Diphenylpropylamine Hydrogen 50 0 2-Fluorophenethylamine
1-Adamantanemethylamine Hydrogen 50 0 2-Fluorophenethylamine
1-(3-Aminopropyl)-2- Hydrogen 50 0 pyrrolidinone (tech)
2-Fluorophenethylamine Decahydroquinoline Hydrogen 50 0
2-Fluorophenethylamine 1-Adamantanamine Hydrogen 50 24.34
2-Fluorophenethylamine 2,4-Dichlorophenethylamine Hydrogen 50 0
2-Fluorophenethylamine Undecylamine Hydrogen 50 0
2-Fluorophenethylamine Dehydroabietylamine Hydrogen 50 0
2-Fluorophenethylamine 2-(1-Cyclohexenyl)ethylamine Methyl 50 0
2-Fluorophenethylamine Cyclooctylamine Methyl 50 5.81
b-Methylphenethylamine 3,3-Diphenylpropylamine Hydrogen 50 0
b-Methylphenethylamine tert-Octylamine Hydrogen 50 0
b-Methylphenethylamine 2-(1-Cyclohexenyl)ethylamine Hydrogen 50 0
b-Methylphenethylamine 2-Amino-2-methyl-1-propanol Hydrogen 50 0
b-Methylphenethylamine 4-Fluorophenethylamine Hydrogen 50 0
b-Methylphenethylamine Geranylamine Hydrogen 50 0
b-Methylphenethylamine 5-Methoxytryptamine Hydrogen 50 0
4-Methoxyphenethylamine 3,3-Diphenylpropylamine Hydrogen 50 0
4-Methoxyphenethylamine 2-Amino-2-methyl-1-propanol Hydrogen 50 0
4-Methoxyphenethylamine 2,4-Dichlorophenethylamine Hydrogen 50 0
4-Methoxyphenethylamine 1-(1-Naphthyl)ethylamine Hydrogen 50 0
L-Methioninol Hexetidine (mixture of isomers) Hydrogen 50 0
Tetrahydrofurfurylamine 1-Adamantanemethylamine Hydrogen 50 0
Tetrahydrofurfurylamine 2-(1-Cyclohexenyl)ethylamine Hydrogen 50 0
Tetrahydrofurfurylamine 4-Fluorophenethylamine Hydrogen 50 0
Tetrahydrofurfurylamine Undecylamine Hydrogen 50 0 Amylamine
1-Adamantanemethylamine Hydrogen 50 0 Amylamine Hexetidine (mixture
of isomers) Hydrogen 50 0 Amylamine Undecylamine Hydrogen 50 0
Amylamine Dehydroabietylamine Hydrogen 50 0 1-Adamantanemethylamine
cis-(-)-Myrtanylamine Methyl 50 0 3-Phenyl-1-propylamine
4-(2-Aminoethyl)morpholine Hydrogen 50 3-Phenyl-1-propylamine
1-(3-Aminopropyl)-2- Hydrogen 50 pyrrolidinone (tech)
3-Phenyl-1-propylamine Veratryl amine Hydrogen 50
3-Phenyl-1-propylamine Aminodiphenylmethane Hydrogen 50
3-Phenyl-1-propylamine 2-(2- Hydrogen 50
Aminomethyl)phenylthio)benzyl alcohol 2,2-Diphenylamine
2-Fluorophenethylamine Hydrogen 50 2,2-Diphenylamine
3,3-Diphenylpropylamine Methyl 50 2,2-Diphenylamine
(+)-Isopinocampheylamine Methyl 50 2,2-Diphenylamine
(+)-Bornylamine Methyl 50 2,2-Diphenylamine Cyclooctylamine Methyl
50 2,2-Diphenylamine (-)-Isopinocampheylamine Methyl 50 3.81
4-(Trifluoromethyl)benzylamine 4-(2-Aminoethyl)morpholine Hydrogen
50 4-(Trifluoromethyl)benzylamine 2-(1-Cyclohexenyl)ethylamine
Hydrogen 50 4-(Trifluoromethyl)benzylamine Hexetidine (mixture of
isomers) Hydrogen 50 4-(Trifluoromethyl)benzylamine
2,4-Dichlorophenethylamine Hydrogen 50
4-(Trifluoromethyl)benzylamine (S)-(-)-Cyclohexylethylamine
Hydrogen 50 Veratryl amine 1-Adamantanemethylamine Hydrogen 50
Veratryl amine 2-(1-Cyclohexenyl)ethylamine Hydrogen 50 Veratryl
amine 4-Fluorophenethylamine Hydrogen 50 Veratryl amine Hexetidine
(mixture of isomers) Hydrogen 50 Veratryl amine
2,4-Dichlorophenethylamine Hydrogen 50 Veratryl amine
(S)-(-)-Cyclohexylethylamine Hydrogen 50 Veratryl amine
Undecylamine Hydrogen 50 Veratryl amine Dehydroabietylamine
Hydrogen 50 Veratryl amine 1-(1-Naphthyl)ethylamine Hydrogen 50
5-Amino-1-pentanol 1-Adamantanemethylamine Hydrogen 50
5-Amino-1-pentanol Dibenzylamine Hydrogen 50 5-Amino-1-pentanol
cis-(-)-Myrtanylamine Hydrogen 50 12.97
2-(1-Cyclohexenyl)ethylamine 2,4-Dimethoxybenzylamine Hydrogen 50
1-Aminomethyl-1- tert-Amylamine Hydrogen 50 cyclohexanol, HCl
1-Aminomethyl-1- 2-(2- Hydrogen 50 cyclohexanol, HCl
Aminomethyl)phenylthio)benzyl alcohol 1-Aminomethyl-1- Undecylamine
Hydrogen 50 cyclohexanol, HCl 1-Aminomethyl-1-
1-(1-Naphthyl)ethylamine Hydrogen 50 cyclohexanol, HCl
3-Fluorobenzylamine tert-Amylamine Hydrogen 50 3-Fluorobenzylamine
Hexetidine (mixture of isomers) Hydrogen 50 3-Fluorobenzylamine
Undecylamine Hydrogen 50 4-Amino-1-butanol Undecylamine Hydrogen 50
4-Amino-1-butanol Dehydroabietylamine Hydrogen 50
2,4-Dimethoxybenzylamine N-Phenylethyldiamine Hydrogen 50
2,4-Dimethoxybenzylamine Aminodiphenylmethane Hydrogen 50
2,4-Dimethoxybenzylamine 4-Phenylbutylamine Hydrogen 50
2,4-Dimethoxybenzylamine 2-Chlorobenzylamine Hydrogen 50
2,4-Dimethoxybenzylamine 2,4-Dichlorophenethylamine Hydrogen 50
2,4-Dimethoxybenzylamine 2-(2-Chlorophenyl)ethylamine Hydrogen 50
2,4-Dimethoxybenzylamine 4- Hydrogen 50
(Trifluoromethoxy)benzylamine 2-Ethoxybenzylamine
Aminodiphenylmethane Hydrogen 50 2-Ethoxybenzylamine
4-Phenylbutylamine Hydrogen 50 2-Ethoxybenzylamine
2-Chlorobenzylamine Hydrogen 50 2-Ethoxybenzylamine 2-Aminoindan,
HCl Hydrogen 50 2-Ethoxybenzylamine 2,5-Dimethoxyphenethylamine
Hydrogen 50 2-Ethoxybenzylamine 4- Hydrogen 50
(Trifluoromethoxy)benzylamine 2-Ethoxybenzylamine
1-(1-Naphthyl)ethylamine Hydrogen 50 cis-(-)-Myrtanylamine
4-(2-Aminoethyl)morpholine Hydrogen 50 cis-(-)-Myrtanylamine
2-Fluorophenethylamine Hydrogen 50 cis-(-)-Myrtanylamine
1-(3-Aminopropyl)-2- Hydrogen 50 pyrrolidinone (tech)
cis-(-)-Myrtanylamine Veratryl amine Hydrogen 50
cis-(-)-Myrtanylamine N-Butylbenzylamine Hydrogen 50
cis-(-)-Myrtanylamine 2,4-Dimethoxybenzylamine Hydrogen 50
cis-(-)-Myrtanylamine 1,2,3,4-Tetrahydropyridoindole Hydrogen 50
cis-(-)-Myrtanylamine 4-Phenylbutylamine Hydrogen 50
cis-(-)-Myrtanylamine 2-(2-Chlorophenyl)ethylamine Hydrogen 50 3.91
cis-(-)-Myrtanylamine 1-(1-Adamantyl)ethylamine, Hydrogen 50 10.85
HCl cis-(-)-Myrtanylamine (R)-(-)-Cyclohexylethylamine Hydrogen 50
5.89 cis-(-)-Myrtanylamine Dehydroabietylamine Hydrogen 50
cis-(-)-Myrtanylamine 1-(1-Naphthyl)ethylamine Hydrogen 50
cis-(-)-Myrtanylamine (+)-Bornylamine Methyl 50 4.04
Cyclooctylamine 4-Methylcyclohexylamine Hydrogen 50 4.55
Cyclooctylamine N-Phenylethyldiamine Hydrogen 50 Cyclooctylamine
4-(Hexacylamino)benzylamine Hydrogen 50 Cyclooctylamine
2,5-Dimethoxyphenethylamine Hydrogen 50 Cyclooctylamine
2,4-Dichlorophenethylamine Hydrogen 50 3.36 Cyclooctylamine
2-(2-Chlorophenyl)ethylamine Hydrogen 50 9.15 Cyclooctylamine
1-(1-Adamantyl)ethylamine, Hydrogen 50 10.62 HCl Cyclooctylamine
(S)-(-)-Cyclohexylethylamine Hydrogen 50 5.85 Cyclooctylamine
(R)-(-)-Cyclohexylethylamine Hydrogen 50 Cyclooctylamine 4-
Hydrogen 50 4.54 (Trifluoromethoxy)benzylamine 2-Adamantanamine,
HCl cis-(-)-Myrtanylamine Hydrogen 50 49.73 4-Methylcyclohexylamine
N-Phenylethyldiamine Hydrogen 50 4-Methylcyclohexylamine
4-Phenylbutylamine Hydrogen 50 4-Fluorobenzylamine
N-Benzyl-2-phenethylamine Hydrogen 50 4-Fluorobenzylamine
Hexetidine (mixture of isomers) Hydrogen 50 4-Fluorobenzylamine
Undecylamine Hydrogen 50 4-Fluorobenzylamine Dehydroabietylamine
Hydrogen 50 trans-2- Hexetidine (mixture of isomers) Hydrogen 50
Phenylcyclopropylamine, HCl trans-2- Undecylamine Hydrogen 50
Phenylcyclopropylamine, HCl trans-2- Dehydroabietylamine Hydrogen
50 Phenylcyclopropylamine, HCl (R)-1-Amino-2-propanol
4-(Hexacylamino)benzylamine Hydrogen 50 (R)-1-Amino-2-propanol
Undecylamine Hydrogen 50 (R)-1-Amino-2-propanol Dehydroabietylamine
Hydrogen 50 I-Leucinol Undecylamine Hydrogen 50
(-)-Isopinocampheylamine 2-Ethoxybenzylamine Hydrogen 50 27.27
(-)-Isopinocampheylamine Hexetidine (mixture of isomers) Hydrogen
50 (-)-Isopinocampheylamine 4-Phenylbutylamine Hydrogen 50
(-)-Isopinocampheylamine Dehydroabietylamine Hydrogen 50
(-)-Isopinocampheylamine 1-(1-Naphthyl)ethylamine Hydrogen 50
Allylamine 3,3-Diphenylpropylamine Hydrogen 50 Allylamine
2-Amino-1-propanol, d,1 Hydrogen 50 Allylamine Undecylamine
Hydrogen 50 3-Amino-1,2-propanediol Dehydroabietylamine Hydrogen 50
3-Ethoxypropylamine 2,2-Diphenylamine Hydrogen 50 95.81
3-Ethoxypropylamine cis-(-)-Myrtanylamine Hydrogen 50
2-Aminoheptane 2-(2- Hydrogen 50 Aminomethyl)phenylthio)benzyl
alcohol 1-Naphthalenemethylamine Geranylamine Hydrogen 50
1-Naphthalenemethylamine Dehydroabietylamine Hydrogen 50
1-Aminopyrrolidine, HCl Hexetidine (mixture of isomers) Hydrogen 50
1-Aminopyrrolidine, HCl Undecylamine Hydrogen 50
1-Aminopyrrolidine, HCl Dehydroabietylamine Hydrogen 50
Ethanolamine 3,3-Diphenylpropylamine Hydrogen 50
3-Methylbenzylamine Geranylamine Hydrogen 50 3-Methylbenzylamine
5-Methoxytryptamine Hydrogen 50 Piperonylamine Aminodiphenylmethane
Hydrogen 50 Piperonylamine 2,4-Dichlorophenethylamine Hydrogen 50
Piperonylamine 2-(2- Hydrogen 50 Aminomethyl)phenylthio)benzyl
alcohol Isopropylamine Dehydroabietylamine Hydrogen 50
4-Fluorophenethylamine 2,4-Dimethoxybenzylamine Hydrogen 50
4-Fluorophenethylamine Aminodiphenylmethane Hydrogen 50
4-Fluorophenethylamine 2-(2- Hydrogen 50
Aminomethyl)phenylthio)benzyl alcohol 4-Chloroamphetamine, HCl
N-Allylcyclopentylamine Hydrogen 50 10.25 4-Chloroamphetamine, HCl
Hexetidine (mixture of isomers) Hydrogen 50 4-Chloroamphetamine,
HCl 4-Phenylbutylamine Hydrogen 50 4-Chloroamphetamine, HCl
2-Methoxyphenethylamine Hydrogen 50 4-Chloroamphetamine, HCl
Undecylamine Hydrogen 50 4-Chloroamphetamine, HCl
Dehydroabietylamine Hydrogen 50 3-Fluorophenethylamine
(-)-Isopinocampheylamine Hydrogen 50 3-Fluorophenethylamine
1-Adamantamine Hydrogen 50 8.59 3-Fluorophenethylamine
4-Phenylbutylamine Hydrogen 50 2-Methylcyclohexylamine (mix
Undecylamine Hydrogen 50 of cis and trans) 2-Methoxyphenethylamine
3,3-Diphenylpropylamine Hydrogen 50 2-Methoxyphenethylamine
(+)-Bornylamine Hydrogen 50 2-Methoxyphenethylamine tert-Octylamine
Hydrogen 50 20.46 2-Methoxyphenethylamine 1-Adamantanemethylamine
Hydrogen 50 2-Methoxyphenethylamine Dibenzylamine Hydrogen 50
2-Methoxyphenethylamine N-Butylbenzylamine Hydrogen 50 5.20
2-Methoxyphenethylamine 1,3,3-Trimethyl-6- Hydrogen 50 8.59
azabicyclo[3.2.1]octane 2-Methoxyphenethylamine
N-Phenylethyldiamine Hydrogen 50 2-Methoxyphenethylamine
2,4-Dichlorophenethylamine Hydrogen 50 2-Methoxyphenethylamine
2-(2-Chlorophenyl)ethylamine Hydrogen 50 2-Methoxyphenethylamine
1-(1-Adamantyl)ethylamine, Hydrogen 50 3.61 HCl 2-Aminoindan, HCl
(+)-Bornylamine Hydrogen 50 2-Aminoindan, HCl Noradamantamine, HCl
Hydrogen 50 7.43 2-(2-Chlorophenyl)ethylamine N-Phenylethyldiamine
Hydrogen 50 2-(2-Chlorophenyl)ethylamine Aminodiphenylmethane
Hydrogen 50 2-(2-Chlorophenyl)ethylamine 2,4-Dichlorophenethylamine
Hydrogen 50 2-(2-Chlorophenyl)ethylamine 1-(1-Adamantyl)ethylamine,
Hydrogen 50 HCl 2-(2-Chlorophenyl)ethylamine Dehydroabietylamine
Hydrogen 50 2-(2- 2-Methoxyphenethylamine Hydrogen 50
Aminomethyl)phenylthio)benzyl alcohol 2-(2-
2,5-Dimethoxyphenethylamine Hydrogen 50
Aminomethyl)phenylthio)benzyl alcohol 2-(2-
2-(2-Chlorophenyl)ethylamine Hydrogen 50
Aminomethyl)phenylthio)benzyl alcohol 2-(2-
1-(1-Adamantyl)ethylamine, Hydrogen 50
Aminomethyl)phenylthio)benzyl HCl alcohol 2-(2- Dehydroabietylamine
Hydrogen 50 Aminomethyl)phenylthio)benzyl alcohol 1-Aminoindan
4-Phenylbutylamine Hydrogen 50 1-Aminoindan
2,4-Dichlorophenethylamine Hydrogen 50 1,3-Dimethylbutylamine
4-Phenylbutylamine Hydrogen 50 (S)-(-)-Cyclohexylethylamine
Aminodiphenylmethane Hydrogen 50 (S)-(-)-Cyclohexylethylamine
4-Phenylbutylamine Hydrogen 50
(S)-(-)-Cyclohexylethylamine 2,4-Dichlorophenethylamine Hydrogen 50
(S)-(-)-Cyclohexylethylamine 1-(1-Adamantyl)ethylamine, Hydrogen 50
HCl (1S,2S)-(+)-2-Amino-3- Dehydroabietylamine Hydrogen 50
methoxy-1-phenyl-1-propanol Octadecylamine 2-Adamantanamine, HCl
Hydrogen 50 3-Hydroxytyramine (1R,2S)-(-)-2-Amino-1,2- Hydrogen 50
diphenylethanol 3-Hydroxytyramine Dehydroabietylamine Hydrogen 50
Geranylamine 3,3-Diphenylpropylamine Hydrogen 50 Geranylamine
N-Phenylethyldiamine Hydrogen 50 Geranylamine Hexetidine (mixture
of isomers) Hydrogen 50 Geranylamine 2-Thiopheneethylamine Hydrogen
50 Geranylamine 2-Methoxyphenethylamine Hydrogen 50 Geranylamine
2,5-Dimethoxyphenethylamine Hydrogen 50 Geranylamine
2,4-Dichlorophenethylamine Hydrogen 50 Geranylamine
2-(2-Chlorophenyl)ethylamine Hydrogen 50 2-Fluorophenethylamine
2,3-Dimethylcyclohexylamine Methyl >50 2.07
4-(Trifluoromethyl)benzylamine 2,3-Dimethylcyclohexylamine Hydrogen
>50- 8.20 4-(Trifluoromethyl)benzylamine 1-Adamantanamine
Hydrogen >50 32.02 5-Aminoquinoline exo-Aminonorbornane Hydrogen
>50 17.87
TABLE-US-00003 TABLE 3 Compounds Synthesized in Larger Quantities
for Further in vitro Evaluations Cmpd # Name Structure Amount, mg
Yields, % 1 N-(4-Methylphenyl)-N'- (furfuryl)ethane-1,2-diamine
##STR00004## 23 25 2 N-(4-Methylphenyl)-N'-
(benzyl)ethane-1,2-diamine ##STR00005## 27 29 3
N-[1-(1,2,3,4-Tetrahydro- naphthalene)-N'-(undecenyl)-
ethane-l,2-diamine ##STR00006## 11 10 4 N-[2-(3,4-Dimethoxyphenyl)-
phenyl)-ethyl-N'-(1- methyladamantyl)-ethane- 1,2-diamine
##STR00007## 13 11 5 N-[2-(3,4-Dimethoxy-
phenyl)ethyl-N'-(norbornyl)- ethane-1,2-diamine ##STR00008## 9 8 6
N-(1-Adamantylmethyl)-N'- (3,3-diphenylpropyl)propane- 1,2-diamine
##STR00009## 55 36 7 N-(1-Adamantylmethyl)-N'-
(3,3-diphenylpropyl)ethane- 1,2-diamine ##STR00010## 28 22 8
N-[2-(Cyclohexen-1-yl)ethyt]- N-(3,3-diphenylpropyl)-
propane-1,2-diamine ##STR00011## 46 37 10
N-(-)-cis-Myrtanyl-N'-(3,3- diphenylpropyl)ethane-1,2- diamine
##STR00012## 14 11 11 N-Cyclooctyl-N'-(3,3-
diphenylpropyl)ethane-1,2- diamine ##STR00013## 22 18 13
N-Allyl-N-cyclopentyl-N'-(3,3- diphenylpropyl)ethane-1,2- diamine
##STR00014## 33 27 14 N-(3,3-Diphenylpropyl)-N'-
exo-(2-norbomy)ethane-1,2- diamine ##STR00015## 17 16 15
1-{2-[N-(3,3-Diphenylpropyl)]- aminoethyl}-3,5-dimethyl- piperidine
##STR00016## 6.2 5 17 N-2-(2-Methoxyphenyl)ethyl-
N'-(3,3-diphenylethyl)ethane- 1,2-diamine ##STR00017## 50 40 21
N-(3,3-Diphenylpropyl)-N'- (1S)-(1-ethylcyclohexane)-
ethane-1,2-diamine ##STR00018## 5 4 22 N-(3,3-Diphenylpropyl)-N'-
(1R)-(1-ethylcyclohexane)- ethane-1,2-diamine ##STR00019## 21 17 23
N-Allyl-N-cyclohexyl-N-(3,3- diphenylpropyl)ethane-1,2- diamine
##STR00020## 6 5 24 N-2-(2-Methoxyphenyl)ethyl-
N'-(4-fluorophenylethyl)- ethane-1,2-diamine ##STR00021## 10 9 27
N-(3-Phenylpropyl)-N'-(1- adamantyl)ethane-1,2- diamine
##STR00022## 11 10 28 N-(3-Phenylpropyl)-N'-(4-
fluorophenyl)ethane-1,2- diamine ##STR00023## 11 10 29
N-(2,2-Diphenylethyl)-N'-(2,3- dimethylcylcohexyl)ethane-
1,2-diamine ##STR00024## 4.5 4 31 N-(2,2-Diphenylethyl)-N'-(1S)-
(1-ethylcyclohexane)-ethane- 1,2-diamine ##STR00025## 24 20 32
N-(2,2-Diphenylethyl)-N'-(R)-(+)- ##STR00026## 58 48 33
N-(2,2-Diphenylethyl)-N'- (1,1,3,3-tetramethylbutyl)-
ethane-1,2-diamine ##STR00027## 11 9 34
N-(2,2-Diphenylethyl)-N'-(1- methyladamantyl)ethane-1,2- diamine
##STR00028## 6.8 6 35 N-(2,2-Diphenylethyl)-N'-
{1,1,3-trimethyl-6-azabicyclo- [3.2.1]octyl}ethane-1,2- diamine
##STR00029## 38 30 36 N-{2-[N'-(2,2-Diphenylethyl)]- aminoethyl}-
decahydroquinoline ##STR00030## 28 24 37
N-(2,2-Diphenylethyl)-N'-(-)- cis-(myrtanyl)ethane-1,2- diamine
##STR00031## 54 38 38 N-(-)-cis-(Myrtanyl)-N'-(2,2-
diphenylethyl)propyl-1,2- diamine ##STR00032## 39 30 40
N-(2,2-Diphenylethyl)-N'-(1R, 2R,3R,5S)-(-)-isopinocam-
pheylethane-1,2-diamine ##STR00033## 33 23 41
N-(-)-cis-(Myrtanyl)-N'-(2,3- dimethylcyclohexyl)ethane -
1,2-diamine ##STR00034## 66 62 42 N-(3,3-Diphenylpropyl)-N'-(-)-
cis-myrtanylethane-1,2- diamine ##STR00035## 11 9 43
N-(-)-cis-Myrtanyl-N'-(1S,2S, 3S,5R)-(+)-
isopinocampheylethane-1,2- diamine ##STR00036## 31 27 47
N-(-)-cis-Myrtanyl-N'-(1R,2R, 3R,5S)-(-) isopinocampheylethane-1,2-
diamine ##STR00037## 42 33 51 N-(Cyclooctyl)-N'-(2,3-
dimethylcyclohexyl)ethane - 1,2-diamine ##STR00038## 5.1 2 52
N-(Cyclooctyl)-N'-(3,3- diphenylpropyl)ethane-1,2- diamine
##STR00039## 20 18 53 N-Cyclooctyl- N'-(1S,2S,3S,
5R)-(+)-isopinocampheyl- ethane-1,2-diamine ##STR00040## 7.4 7 54
N-Cyclooctyl-N'-(R)-(+)- bornylethane-1,2-diamine ##STR00041## 17
16 55 N-(Cyclooctyl)-N'-(1- methyladamantyl)ethane-1,2- diamine
##STR00042## 7 6 56 N-(Cyclooctyl)-N'-(2S)-[2-(1-
hydroxybutyl)]ethane-1,2- diamine ##STR00043## 1.1 1 57
N-(-)-cis-Myrtanyl-N'- (cyclooctyl)ethane-1,2- diamine ##STR00044##
18 18 58 N-(Cyclooctyl)-N'-(2- adamantyl)ethane-1,2- diamine
##STR00045## 25 23 59 N-(Cyclooctyl)-N'-(1R,2R, 3R,5S)-(-)-
isopinocampheylethane-1,2- diamine ##STR00046## 15 14 61
N-(Cyclooctyl)-N'-[1-ethyl-(1- naphthyl)]ethane-1,2-diamine
##STR00047## 16 14 62 N-(-)-cis-Myrtanyl-N'-(1S)-(1-
ethylcyclohexane)ethane-1,2-diamine ##STR00048## 48 46 63
N-(Cyclooctyl)-N'-trans-(2- phenylcyclopropyl)ethane- 1,2-diamine
##STR00049## 47 46 64 N-(2-Adamantyl)-N'-trans-(2-
phenylcyclopropyl)ethane- 1,2-diamine ##STR00050## 49 46 65
N-(1-Adamantyl)-N'-trans-(2- phenylcyclopropyl)ethane- 1,2-diamine
##STR00051## 18 16 66 N-(3,3-Diphenylpropyl)-N'- (1R,2R,3R,5S)-(-)-
isopinocampheylethane-1,2- diamine ##STR00052## 2.3 2 68
N-(+/-)-[2-(1-Hydroxybutyl)]- N''-(1R,2R,3R,5S)-(-)-
isopinocampheylethane-1,2- diamine ##STR00053## 0.8 1 71
N-(1,1-Diphenylmethyl)-N'- (1R,2R,3R,5S)-(-)-
isopinocampheylethane-1,2- diamine ##STR00054## 2.9 2 73
N-(2-Adamantyl)-N'[2-(2- methoxyphenyl)ethyl]ethane- 1,2-diamine
##STR00055## 21 19 76 N-Allyl-N-cyclopentyl-N'-[2-(2-
methoxyphenyl)ethyl]ethane- 1,2-diamine ##STR00056## 8 7 77
N-(1,1-Diphenylmethyl)-N'-[2- (2-methoxyphenyl)-
ethyl]ethane-1,2-diamine ##STR00057## 32 27 78
N-2-Adamantyl-N'-2,3- dihydro-1H-inden-2-yl- ethane-1,2-diamine
##STR00058## 4.3 3 79 N-[2-(2,5-Dimethoxyphenyl)-
ethyl]-N'-(R)-(+)- bornylethane-1,2-diamine ##STR00059## 59 49 103
N,N'-Bis(cyclooctyl)ethane- 1,2-diamine ##STR00060## 6.3 4 107
N-(2,2-Diphenylethyl)-N-(3- ethoxypropyl)ethane-1,2- diamine
##STR00061## 58 52 109 N-Geranyl-N'-(2- adamanthyl)ethane-1,2-
diamine ##STR00062## 27 24 111 N-[2-(N'-Geranyl)aminoethyl]-
2-ethylpiperidine ##STR00063## 24 24 116 N-Geranyl-N'-allyl-N'-
(cyclopentyl)ethane-1,2- diamine ##STR00064## 45 42 117
N-Geranyl-N'-(1,1-diphenyl- methyl)ethane-1,2-diamine ##STR00065##
24 20 118 N-2-(2-Chlorophenyl)ethyl-N'-
allyl-N'-(cyclopentyl)ethane- 1,2-diamine ##STR00066## 6.4 6 119
N-2-(2-Chlorophenyl)ethyl- N'-[2-(3-fluorophenyl)-
ethyl]ethane-1,2-diamine ##STR00067## 30 27 125 N,N'-bis-(-)-cis-
Myrtanylpropane-1,2-diamine ##STR00068## 41 35 134
N-[2-(N'-2,2-Diphenylethyl)- aminoethyl]-(-)-3,4-
dihydroxynorephedrine ##STR00069## 20 15 151
N-[2-(2-Methoxy)phenylethyl]- N'-(1R,2R,3R,5S)-(-)-
isopinocampheyl-ethane-1,2- diamine ##STR00070## 67 60 164
N.sup.1-[2-(4-fluorophenyl)ethyl]- N.sup.2-[2-(4-Methoxy)
phenylethyl)-1-phenylethane- 1,2-diamine ##STR00071## 94 73 165
N1-[2-(4-fluorophenyl)ethyl]- N2-(3-Phenylpropyl)-1-
phenylethane-1,2-diamine ##STR00072## 23 19
The present invention is also directed to a new library of diamine
compounds useful against infectious disease. To further enhance the
structural diversity of prior diamine compounds, a synthetic scheme
to incorporate amino acids into a bridging linker between the two
amine components has been developed. The use of amino acids allowed
for diverse linker elements, as well as chirality see FIG. 42 for
representative examples. The diamine compounds were prepared on
mmol scale in 96-well format in pools of 10 compounds per well (for
the vast majority of the plates). Table 25 (FIG. 43) summarizes
data for the synthesized plates.
The reaction scheme followed is shown in FIG. 44.
Solid phase syntheses using Rink resin. Twenty one 96-well plates
have been prepared. Six-step synthetic route starting from the Rink
resin similar to what that had been used to create our first
100,000 compound library (Scheme 1, FIG. 41), was applied to make
targeted diamines (Scheme 5, FIG. 44). Overall, all steps of these
schemes are similar, except one (step 4) when formation of the
second amino functionality occurs. In Scheme 1, the second amine is
introduced into the molecule as a whole synthon via nucleophilic
displacement of C1-function of the linker, while in the Scheme 5,
it proceeds through modification of the existing amino moiety by
carbonyl compounds.
Attachment of the first amine to the support was done according to
the Garigipati protocol. Rink acid resin (Novabiochem) was
converted into the Rink-chloride upon treatment with
triphenylphosphine and dichloroethane in THF. This activated resin
was then loaded by addition of an amine N1 in presence of Hunig's
base in dichloroethane. The amine N1 includes, but is not limited
to, alkyl and aryl primary amines. Out of 177 primary amines that
had been previously used as N1 for 100,000 library preparation,
only 30 were selected in this Scheme, based upon in vitro activity
data of their ethylenediamine derivatives (from the previous
.about.100K library) as well as structural diversity (FIGS. 45 and
46).
On the next step, the acylation reaction was accomplished via
peptide coupling with FMOC protected amino acids in presence of
HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium
hexafluorophosphate) and EtN(iso-Pr).sub.2 in DCM/DMF mixture at
room temperature. The reaction was done twice to improve product
yields. The list of the amino acids used to create this library is
shown in the Table 26 (FIG. 47).
Deprotection (removal of the FMOC group) was carried out by
reaction with piperidine at room temperature. Derivatization of the
amino group was achieved by reductive alkylation with various
carbonyl compounds, such as aldehydes, ketones, and carboxylic
acids, in the presence of NaBCNH.sub.3 at room temperature for
72-96 h. The selection of the carbonyl compounds was made so that
the final diamine products would carry the same or similar types of
substituents that had been observed in the hit compounds generated
from the previous library of ethambutol analogs, as well as
structural diversity (FIG. 48). A complete list of the carbonyl
compounds used is shown in Table 27 (FIG. 49).
Reduction of the aminoethyleneamides into corresponding diamines
was carried out using the soluble reducing reagent 65+w % Red-A1 at
room temperature. Cleavage of the products from the resin was
achieved with a 10% solution of trifluoroacetic acid in
dichloromethane resulting in the formation of TFA salts of the
diamines.
For library production the first three steps of the synthetic
scheme (resin activation, amine loading, and acylation) were
carried out using a Quest 210 Synthesizer on scale of 0.1-0.15 g of
resin per tube. Following the acylation, formed resins were
thoroughly washed, dried, and then groups of ten resins were pooled
together. A small amount of each resin (.about.0.05 g) was archived
prior to pooling to facilitate re-synthesis and deconvolution of
actives.
Deprotection of the FMOC group, addition of the carbonyl component,
reduction, and cleavage were carried out in 96-well reaction blocks
using the Combiclamps system by Whatman Polyfiltronics or the
FlexChem system by Robbins Scientific. A suspension of the pooled
resins in 2:1 mixture of DCM/THF was evenly distributed into one
reaction plate resulting in approximately 10 mg of the resin per
well. The 96 diverse carbonyl compounds were arrayed in one 96-well
plate template and added, one carbonyl compound per well, to each
individual pool of ten resins, resulting in an anticipated 960
diamines produced per plate. Reduction was carried out in the same
format and cleavage and filtering into storage plates was followed
by evaporation of the TFA prior to biological assay.
Quality assessment of the prepared compounds was done by
Electrospray Ionization mass spectrometry using two randomly
selected rows (16 samples) per plate, 17% of the total number.
Successful production of a compound was based on an appearance of a
molecular ion of the calculated mass. Depending on the amino acid
that had been used for the synthesis, the percentage of the
predicted ions were observed, and therefore the predicted compounds
were formed, varied from 5-60% (Table 25, FIG. 43). Based on MS
analysis, out of targeted 20,000 compounds, 4,500 diamines were
actually formed.
Formulations
Therapeutics, including compositions containing the substituted
ethylene diamine compounds of the present invention, can be
prepared in physiologically acceptable formulations, such as in
pharmaceutically acceptable carriers, using known techniques. For
example, a substituted ethylene diamine compound is combined with a
pharmaceutically acceptable excipient to form a therapeutic
composition.
The compositions of the present invention may be administered in
the form of a solid, liquid or aerosol. Examples of solid
compositions include pills, creams, soaps and implantable dosage
units. Pills may be administered orally. Therapeutic creams and
anti-mycobacteria soaps may be administered topically. Implantable
dosage units may be administered locally, for example, in the
lungs, or may be implanted for systematic release of the
therapeutic composition, for example, subcutaneously. Examples of
liquid compositions include formulations adapted for injection
intramuscularly, subcutaneously, intravenously, intraarterially,
and formulations for topical and intraocular administration.
Examples of aerosol formulations include inhaler formulations for
administration to the lungs.
A sustained release matrix, as used herein, is a matrix made of
materials, usually polymers, which are degradable by enzymatic or
acid/base hydrolysis, or by dissolution. Once inserted into the
body, the matrix is acted upon by enzymes and body fluids. The
sustained release matrix is chosen desirably from biocompatible
materials, including, but not limited to, liposomes, polylactides,
polyglycolide (polymer of glycolic acid), polylactide co-glycolide
(coplymers of lactic acid and glycolic acid), polyanhydrides,
poly(ortho)esters, polypeptides, hyaluronic acid, collagen,
chondroitin sulfate, carboxylic acids, fatty acids, phospholipds,
polysaccharides, nucleic acids, polyamino acids, amino acids such
as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl
propylene, polyvinylpyrrolidone and silicone. A preferred
biodegradable matrix is a matrix of one of either polylactide,
polyglycolide, or polylactide co-glycolide.
The dosage of the composition will depend on the condition being
treated, the particular composition used, and other clinical
factors, such as weight and condition of the patient, and the route
of administration. A suitable dosage may range from 100 to 0.1
mg/kg. A more preferred dosage may range from 50 to 0.2 mg/kg. A
more preferred dosage may range from 25 to 0.5 mg/kg. Tablets or
other forms of media may contain from 1 to 1000 mg of the
substituted ethylene diamine. Dosage ranges and schedules of
administration similar to ethambutol or other anti-tuberculosis
drugs may be used.
The composition may be administered in combination with other
compositions and procedures for the treatment of other disorders
occurring in combination with mycobacterial disease. For example,
tuberculosis frequently occurs as a secondary complication
associated with acquired immunodeficiency syndrome (AIDS). Patients
undergoing AIDS treatment, which includes procedures such as
surgery, radiation or chemotherapy, may benefit from the
therapeutic methods and compositions described herein.
The following specific examples will illustrate the invention as it
applies to the particular synthesis of the substituted ethylene
diamine compounds, and the in vitro and in vivo suppression of the
growth of colonies of M. tuberculosis. In additiona, the teachings
of R. Lee et al. J. Comb. Chem 2003, 5, 172-187 are hereby
incorporated by reference in their entirety. It will be appreciated
that other examples, including minor variations in chemical
procedures, will be apparent to those skilled in the art, and that
the invention is not limited to these specific illustrated
examples.
EXAMPLE I
Generating the Ethylene Diamine Library
The Rink-acid resin was obtained from NOVABIOCHEM.RTM. Inc., San
Diego, Calif. Solvents: acetonitrile, dichloromethane,
dimethylformamide, ethylenedichloride, methanol and tetrahydrofuran
were purchased from ALDRICH.RTM., Milwaukee, Wis., and used as
received. All other reagents were purchased from
SIGMA-ALDRICH.RTM., West Monroe Highland, Ill. Solid phase
syntheses were performed on a QUEST.RTM. 210 Synthesizer, from
ARGONAUT TECHNOLOGIES.RTM., Foster City, Calif., with the aid of
combinatorial chemistry equipment, from WHATMAN.RTM.
POLYFILTRONICS.RTM. (Kent, England; Rockland, Mass.) and ROBBINS
SCIENTIFIC.RTM., Sunnyvale, Calif. Evaporation of solvents was done
using SPEEDVAC.RTM. AES, from SAVANT.RTM., Holbrook, N.Y. All
necessary chromatographic separations were performed on a WATERS'
ALLIANCE HT SYSTEM.RTM., Milford, Mass. Analytical thin-layer
chromatography was performed on MERCK.RTM. silica gel 60F.sub.254
plates, purchased from SIGMA-ALDRICH.RTM., West Monroe Highland,
Ill.
The activation of the Rink-acid resin, the addition of the first
amine, and the acylation step were carried out in 10 ml tubes using
the QUEST.RTM. 210 Synthesizer. The addition of the second amine,
the reduction with Red-AL, and the cleavage from the solid support
were carried out in 96-deep (2 ml) well, chemically resistant
plates.
A. Activation of the Rink-Acid Resin
The Rink-acid resin had a coverage of 0.43-0.63 mmol of linker per
gram resin. Four to five grams of this resin were suspended in 80
ml of a 2:1 mixture of dichloromethane and tetrahydrofuran (THF),
and distributed into ten, 10 ml tubes, with 8 ml of resin
suspension per tube. Each suspension was filtered and washed twice
with THF. A solution of triphenylphosphine (3.80 g, 14.5 mmol) in
30 ml of THF was prepared, and 3 ml of this solution was added to
each tube, followed by the addition of 3 ml of a solution of
hexachloroethane in THF (3.39 g/14.3 mmol hexachloroethane in 30 ml
THF). After agitation for six hours at room temperature, each
activated resin was washed twice with THF and twice with
dichloromethane.
B. Addition of the First Amine
Each tube, containing the activated rink resin, was charged with 3
ml of dichloroethane, 0.3 ml (1.74 mmol)
N.sub.1N-diisopropylethylamine (EtN(iPr).sub.2) and the
corresponding amine (around 1 mmol). If the selected amine was a
solid at room temperature, it was added as a solution, or a
suspension in DMF. Enough dichloroethane was added to each tube for
a final volume of 8 ml. The reaction mixture was heated at
45.degree. C. for 6-8 hours. The resins were filtered, washed with
a 2:1 mixture of dichloromethane and methanol (1.times.8 ml), then
with methanol (2.times.8 ml), and then dried under argon for 10
minutes.
C. Acylation with the Halo-Acylchloride
a. Acylation with Chloroacetyl Chloride. Each resin was prewashed
with TBF (2.times.8 ml), and then charged with TBF (8 ml), pyridine
(0.3 ml, 3.67 mmole) and chloroacetyl chloride (0.25 ml, 2.5
mmole). The reaction mixture was stirred for 8 hours at 45.degree.
C., and then for 6-8 hours at room temperature. Each resin was
filtered, washed with a 2:1 mixture of dichloromethane/methanol
(1.times.8 ml), methanol (2.times.8 ml) and TBF (2.times.8 ml). The
acylation was repeated using the same loading of reagents, but a
shorter reaction time of 4 hours at 45.degree. C., and 2 hours at
room temperature. Each resin was then filtered, washed with a 2:1
mixture of dichloromethane and methanol (1.times.8 ml), and then
with methanol (3.times.8 ml). Each resin was dried under argon for
10 minutes. Each resin was then transferred into a vial and dried
in a desiccator under vacuum for 1 hour.
b. Acylation with .alpha.-Phenyl-.alpha.-Chloroacetyl Chloride. The
same procedure set out for the acylation with chloroacetyl chloride
was used. A 2.5 mmol excess of c phenyl-.alpha.-chloroacetyl
chloride, relative to mmol amount of linker in the rink-acid resin,
was used.
c. Acylation with .alpha.-Halo-.alpha.-Methyl;
.alpha.-Halo-.alpha.-Ethyl and .alpha.-Halo-.alpha.-Butylacetyl
Bromide. A 1:1:1 mixture (by volume) of the .alpha.-bromoproponyl
bromide (R.sub.4=Me), .alpha.-bromobutyryl bromide (R.sub.4=Et),
and .alpha.-bromohexanoyl bromide (=Bu) was used to give a molar
ratio of 0.52:0.56:0.42 (in mmols). This resulted in a molar excess
of 1.65, 1.75 and 1.31, respectively, if the original coverage of
the resin was 0.63 mmol/g (0.5 g resin per tube), and 2.4, 2.6 and
1.9 if the original coverage of the resins was 0.43 mmol/g (0.5 g
resin per tube).
d. Acylation with .alpha.-Chloro-.alpha.-Methyl Acetic acid. Each
resin was prewashed with dichloromethane. Each tube was charged
with 3 ml of a solution of PyBrop (0.29 g, 0.62 mmole) in
dichloromethane, a solution of the
.alpha.-chloro-.alpha.-methylacetic acid (0.095 g, 0.62 mmole) in 3
ml of DMF, and EtN(iPr).sub.2 (0.2 ml, 1.2 mmole). Each reaction
mixture was allowed to react for 16-18 hours at room temperature.
Each resin was then filtered, washed with dichcloromethane
(2.times.8 ml) and methanol (2.times.8 ml), and the acylation was
repeated. Each resin was then filtered, washed with dichloromethane
(2.times.8 ml), methanol (3.times.8 ml), and dried under argon for
about 10 minutes. Each resin was transferred into a vial, and dried
in a desiccator under vacuum for one hour.
D. Addition of the Second Amine
Ten, or thirty prepared .alpha.-haloacetyl amide resins from the
first three steps were pooled together, leaving 0.05-0.10 gram of
each individual resin for necessary deconvolutions. A suspension of
the pooled resin mixture in 100 ml of a 2:1 mixture of
dichloromethane and THF was distributed into one, two or three,
96-well reaction plates. For one reaction plate, 1.7 to 2.0 grams
of resin were used. For two reaction plates, 3.0 to 3.3 grams of
resin were used, and for three reaction plates, 4.7 to 5.0 grams of
resin were used. The distributed suspension was then filtered using
a filtration manifold, a small lightweight manifold that is
generally used for drawing solvents and reagents from the chambers
of the 96-well reaction plates. The reaction plates were
transferred into COMBICLAMPS.RTM. (Huntington, West Va.), and 10%
EtN(iPr).sub.2 in DMF was added at 0.2 ml per well (0.21 mmole of
EtN(iPr).sub.2 per well), followed by the addition of a 1.0M
solution of the appropriate amine from the corresponding master
plate, 0.1 ml per well (0.1 mmole amine per well). The
COMBICLAMPS.RTM. are used to accommodate 96-well reaction plates
during synthesis, allowing for the addition of reagents into the
plates, and a proper sealing that maintains reagents and solvents
for hours at elevated temperatures. These clamps consist of a top
and bottom cover provided with changeable, chemically resistant
sealing gaskets. They are designed to accommodate 96-well reaction
plates between the top and bottom covers. The reaction plates were
sealed and kept in an oven at 70-75.degree. C. for 16 hours. After
cooling to room temperature, the resins were filtered, washed with
a 1:1 mixture of DCM/methanol (1.times.1 ml), methanol (2.times.1
ml), and then dried in a desiccator under vacuum for 2 hours.
E. Reduction with Red-A1
The reaction plates were placed into COMBICLAMPS.RTM.. A 1:6
mixture of Red-A1 (65+w % in toluene) and THF was added, at 0.6 ml
per well (0.28 mmole of Red-A1 per well), and allowed to react for
4 hours. Each resin was then filtered, washed with THF (2.times.1
ml), and methanol (3.times.1 ml). The addition of methanol should
proceed with caution. Each resin was then dried under vacuum.
F. Cleavage of Final Ethylene Diamine Compound
This step was carried out using a cleavage manifold, a Teflon
coated aluminum, filter/collection vacuum manifold, designed for
recovering cleavage products from the reaction plates into
collection plates. The manifold is designed to ensure that the
filtrate from each well is directed to a corresponding well in a
receiving 96-well collection plate. The reaction plates (placed on
the top of the collection plates in this manifold) were charged
with a 10:85:5 mixture of TFA, dichloromethane, and methanol (0.5
ml of mixture per well). After fifteen minutes, the solutions were
filtered and collected into proper wells on the collection plates.
The procedure was repeated. Solvents were evaporated on a SPEED
VAC.RTM., Holbrook, N.Y., and the residual samples (TFA salts) were
tested without further purification.
EXAMPLE II
Deconvolution Example
Deconvolution of the active wells was performed by re-synthesis of
discrete compounds, from the archived .alpha.-haloacetyl amide
resins (10 resins, 0.05-0.10 g each), which were set aside at the
end of the acylation step before the pooling. Each resin was
assigned a discrete column (1, or 2, or 3, etc., see the template)
in a 96 well filterplate, and was divided between X rows (A, B, C,
etc), where X is the number of hits discovered in the original
screening plate. To each well, in a row, a selected N2
(R.sub.3R.sub.2NH) hit amine (0.1 mmol), DMF (180 ml) and
EtNiPr.sub.2 (20 ml) were added: the first selected amine was added
to the resins in the row "A", the second amine--to the resins in
the row "B", the third amine--to the resins in the row "C", etc. A
lay-out of a representative 96-well filter plate is shown in Table
4.
The reaction plates were sealed and kept in an oven at
70-75.degree. C. for 16 hours. After cooling to room temperature,
the resins were filtered, washed with a 1:1 mixture of DCM and
methanol (1.times.1 ml), methanol (2.times.1 ml), and dried in
desiccator under vacuum for 2 h. Reduction and cleavage were
performed according to steps 5 and 6 in the original synthetic
protocol. The product wells from the cleavage were analyzed by
ESI-MS (Electro Spray Ionization Mass Spectroscopy) to ensure the
identity of the actives, and were tested in the same Luc and MIC
assays.
TABLE-US-00004 TABLE 4 Lay-Out of Representative 96-Well Filter
Plate A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 Selected amine N2, Added to
A1-A10 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Selected amine N2, Added to
B1-B10 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Selected amine N2, Added to
C1-C10 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 Selected amine N2, Added to
D1-D10 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 Selected amine N2, Added to
E1-E10 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 Selected amine N2, Added to
F1-F10 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 Selected amine N2, Added to
G1-G10 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 Selected amine N2, Added to
H1-H10 Resin Resin Resin Resin Resin Resin Resin Resin Resin Resin
*X* selected #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 Amines N2 to be added
on the step 4 Individual resins ##1-10, preloaded with proper amine
N1.
EXAMPLE III
Solid-Phase Synthesis of Selected Substituted Ethylenediamine
Compounds Using the QUEST.RTM. 210 Synthesizer
The solid-phase protocol described above in Example I was applied
to the scaled-up synthesis of the selected substituted ethylene
diamine compounds. Here, all reaction steps, from the activation of
the Rink-acid resin to the cleavage of the final product, were
carried out using the QUEST.RTM. instrument only, which allowed for
the simultaneous syntheses of twenty parallel reactions.
Purification of all crude samples was done by HPLC to yield
desirable products in purity greater than 90%. Table 3 lists the
scale-ups of substituted ethylene diamines. Here, the synthesis of
one of the active compounds,
N-Geranyl-N'-(2-adamanthyl)ethane-1,2-diamine is described below as
an example.
The Preparation of N-Geranyl-N'-(2-adamanthyl)ethane-1,2-diamine
(compound 109) is set forth in FIG. 12.
##STR00073## 1. Activation of the Rink-acid resin. Synthesis of
Rink-C1 resin. Rink-acid resin, coverage (linker) of 0.43 to 0.63
mmol/g (0.8 g, 0.5 mmol), was placed into one of the 10 ml tubes of
QUEST.RTM. 210 Synthesizer, and washed twice with THF. A solution
of triphenylphosphine (0.380 g, 1.45 mmol) in THF (3 ml) was added,
followed by the addition of a solution of hexachloroethane (0.4 g,
1.43 mmol) in THF (3 rml). THF was added up to the volume of the
tube (approximately 2 ml). After 6 hours, the resin was filtered,
washed with THF (2.times.8 nml) and dichloromethane (2.times.8 ml).
2. Addition of the first amine. Synthesis of resin attached
geranylamine. The tube with activated resin was charged with 3 ml
of dichloroethane, EtN(iPr).sub.2, (0.3 ml, 1.74 mmol), and
geranylamine (0.230 g, 1.5 mmol). Dichloroethane was added to a
volume of 8 ml. The reaction was carried for 8 hours at 45.degree.
C., and for 6-8 hours at room temperature. Geranylamine loaded
resin was filtered, washed with a 2:1 mixture of dichloromethane
and methanol (1.times.8 ml), then with methanol (2.times.8 ml), and
suck dried for 10 minutes under argon. 3. Acylation with
chloroacetyl chloride. Synthesis of resin attached
N-Geranyl-.alpha.-chloroacetamide. The resin was prewashed with THF
(2.times.8 ml). The tube was charged with 8 ml of THF, pyridine
(0.3 ml, 3.67 mmol), and chloroacetyl chloride (0.2 ml, 2.5 mmol),
and allowed to stir for 8 h at 45.degree. C., and 6-8 h at room
temperature (RT). After the reaction was complete, the resin was
filtered, washed with a 2:1 mixture of dichloromethane and methanol
(1.times.8 ml), methanol (2.times.8 ml), and THF, and the acylation
was repeated using the same loads of the reagents, but shorter
reaction time: 4 hours at 45.degree. C. and 2 hours at room
temperature. At the end, the .alpha.-chloroacetamide loaded resin
was filtered, washed with a 2:1 mixture of dichloromethane and
methanol (1.times.8 ml), methanol (3.times.8 ml), and suck dried
for 15 min under argon. 4. Addition of the second amine. Synthesis
of resin attached N-Geranyl-N'-(2-adamantyl)acetamide. The tube
with the resin was charged with DMF (3 ml) and EtN(ipr).sub.2 (0.6
ml, 4.4 mmol), followed by the addition of a suspension of
2-adamantamine hydrochloride (2.0 g, 1.1 mmol) in DMF (4 ml), and
was allowed to stir at 70-75.degree. C. for 16 hours. After cooling
down to the room temperature, the resin was filtered, washed with a
1:1 mixture of DCM and methanol (1.times.8 ml), methanol (2.times.8
ml), and suck dried for 15 minutes under argon. 5. Reduction with
Red-A1. Synthesis of resin attached
N-Geranyl-N'-(2-adamantyl)ethane-1,2-diamine. The resultant resin
was suspended in anhydrous TIF (3 ml) in a tube, and stirred for 15
min. Commercially available Red-A1, 65+w % in toluene, was added
(2.0 ml, 6.4 mmol), followed by addition of 2-3 ml of anhydrous THF
(to fill up the volume of the tube). The mixture was allowed to
react for 4 hours. After the reaction, the resin was filtered,
washed with THF (1.times.8 ml), a 1:1 mixture of THF and methanol
(1.times.8 ml) (addition of MeOH should proceed with caution),
methanol (3.times.8 ml), and then dried. 6. Cleavage from the resin
and purification. Synthesis of
N-Geranyl-N'-(2-adamanthyl)ethane-1,2-diamine acetate. For this
last step of the synthesis, the tube with the resin was charged
with a 10:90 mixture of TFA and dichloromethane, and the formed
bright red suspension was allowed to stir for 30 min. After
addition of MeOH (0.5 ml), the colorless suspension was filtered,
and the filtrate was collected into a proper tube. The procedure
was repeated, and solvents were evaporated on a SPEEDVAC.RTM.. Half
of the amount of crude
N-Geranyl-N'-(2-adamanthyl)ethane-1,2-diamine (in a form of
trifluoroacetate salt) was purified by HPLC using following
conditions: column C18, flow 4 ml/min, 30 min run, gradient
starting with 5% AcOH/MeOH (100%) finishing up with acetonitrile
(100%). Obtained: 27 mg of
N-Geranyl-N'-(2-adamanthyl)ethane-1,2-diamine diacetate, 24% yield,
98% purity by NMR.
EXAMPLE IV
Representative Solution Phase Synthesis of the Active Compounds
Preparation of
N-(Cyclooctyl)-N'-(1R,2R,3R,5S)-(-)-isopinocampheylethane-1,2-diamine
as hydrochloride (compound 59) is set forth in FIG. 13.
##STR00074##
Bromocyclooctylacetylamide. To a mixture of cyclooctylamine (3.3 g,
0.026 mol) and pyridine (2.42 g, 0.031 mmol) in anhydrous THF (80
ml) at 0.degree. C. was added dropwise, via syringe,
bromoacetylbromide (5.78 g, 0.029 mol). The reaction temperature
was maintained by an ice bath. The reaction mixture was allowed
gradually to warm up to room temperature, and was stirred at room
temperature for 1 hour. The precipitate was removed by filtration,
washed with ethyl ether (1.times.30 ml), and the filtrate was
concentrated to dryness on a rotory evaporator.
Bromocyclooctylacetylamide was forwarded to the second step without
additional purification.
N-(Cyclooctyl)-N'-(1R,2R,3R,5S)-(-)-isopinocampheyl-1-carbonylethane-1,2--
diamine. To a solution of the bromocyclooctylacetylamide in DMF (60
ml) were added Hunig's base (4.64 g, 0.036 mol) and
(1R,2R,3R,5S)-(-)-isopinocampheylamine (4.5 g, 0.029 mol), and the
reaction mixture was stirred at 80.degree. C. for 16 hours. After
cooling off to the room temperature, the reaction mixture was
diluted with 150 ml of ethyl ether, and washed with 1M NaOH
solution (2.times.50 ml). The organic layer was washed with brine
(1.times.50 ml), dried over MgSO.sub.4, and concentrated to dryness
on the rotory evaporator. The residue (11.04 g) as brown oil was
purified on COMBIFLASK.RTM. (Isco, Lincoln, Nebr., USA), using
Silicagel catridges commercially available from BIOTAGES (Biotage,
Inc. of Dyax Corp, Va, USA), and the following mobile phase
gradient: 30 min run, starting with DCM, 100%, and finishing up
with a mixture DCM:MeOH:NH.sub.4OH (600:400:10). The final product
(7.29 g) was obtained as a brown oil; 76% yield, purity 90%.
N-(Cyclooctyl)-N'-(1R,2R,3R,5S)-(-)-isopinocampheylethane-1,2-diamine.
To a solution of the amide, from previous step, in anhydrous ThF
(160 ml), was added dropwise via syringe commercially available
(SIGMA-ALDRICH.RTM.) Red-A1, as 65 wt % solution in THF (28 ml,
0.09 mol). The reaction mixture was stirred at reflux for 20 hours.
After cooling down to the room temperature, the reaction mixture
was poured into 1.5M NaOH (200 ml), and extracted with ethyl ether
(2.times.100 ml). The organic layer was washed with brine
(1.times.100 ml), dried over MgSO.sub.4, and evaporated to dryness
on the rotory evaporator to yield 7.2 g of a crude product, as a
brown oil. Chromatographic purification of the crude using the same
equipment and conditions as for the previous step, gave 3.5 g of
the diamine. The diamine was treated with 2.0M solution of HCl in
ethyl ether (25 ml), and kept in a refrigerator overnight. A dark
yellow solid (4.2 g) formed, and was filtered off, and
recrystallized from MeOH and ethyl ether to yield 1.5 g of the
diamine as an HCl salt (of purity greater than 98%, NMR and MS are
available), 19% overall yield.
EXAMPLE V
Mass Spectroscopy Analysis
Mass spectra data were obtained by Elecrospray Ionization technique
on a PERKIN ELMER.RTM./SCIEX.RTM., API-300, TQMS with an
autosampler, manufactured by SCIEX.RTM., Toronto, Canada.
A. Library of Substituted Ethylenediamines
Mass spectroscopy served as a means for monitoring the reaction
results of the library of ethylenediamines. Mass spectroscopy was
done on two randomly selected rows (24 samples) per reaction plate,
for roughly 28,000 compounds in pool of 10 or 30 compounds per
well. Thus, if ten compounds per well were synthesized, the mass
spectra for each well should contain ten signals, correlating with
the proper molecular ions for each compound. The presence or
absence of a particular signal indicated the feasibility of the
particular synthesis. Based on the mass spectral data, and on a
general analysis of the reactivity of the various amines, it is
estimated that 67,000 compounds were formed out of 112,000
compounds.
FIG. 14 is a representative mass spec profile for one sample well.
Mass spectra for a representative ethylene diamine compound is
shown in FIG. 15. Tables 5 to 8, below, list illustrative examples
of mass spec data for representative reaction wells, with each well
containing ten substituted ethylene diamines.
TABLE-US-00005 TABLE 5 ILLUSTRATIVE EXAMPLES OF MASS SPEC DATA FOR
REPRESENTATIVE ETHYLENEDIAMINES (TEN COMPOUNDS PER WELL).
R.sub.2R.sub.3NH in the 2.sup.nd position R.sub.1NH.sub.2 in the
1.sup.st position (pool of 10 (from the master plate of the [M +
1].sup.+ of the product resins) amines)
R.sub.1NHCH.sub.2CH.sub.2NR.sub.2R.sub.3 Plate # 4-034-2, well D10
1-(2-Aminoethyl)piperidine 2-Aminoheptane 270 absent Phenethylamine
263 4-(2-Aminoethyl)morpholine 272 absent Tryptamine 302
Cyclohexylamine 241 Exo-2-Aminonorbomane 253 Benzylamine 249
2-Fluorophenethylamine 281 ?-Methylphenethylamine 277
4-Methoxyphenethylamine 293 Plate # 4-56-1, well C4
4-Methylbenzylamine exo-2-Aminonorbornane 259 Cyclopentylamine 223
2-(Aminomethyl)piperidine 246 low intensity Furfurylamine 235
3,4,5-Trimethoxybenzylamine 335 1-Methyl-3-phenylpropylamine 287
Cylcobutylamine 209 1,2,3,4-Tetrahydro-1-naphthylamine 258
2,3-Dimethylcyclohexylamine 265 2-Amino-1-butanol 227 low intensity
Plate # 4-44-2, well G1 Veratrylamine 4-Fluorophenethylamine 333
2-(1-Cyclohexenyl)ethylamine 291 5-Aminoquinolone 310 absent
1-(1-Naphthyl)ethylamine 337 absent 1-Aminopiperidine 266
3-Fluorobenzylamine 291 2,4-Dimethoxybenzylamine 333
3-Amino-1,2,4-triazine 262 absent 2-Ethoxybenzylamine 317
4-(3-Aminopropyl)morpholine 310 absent
TABLE-US-00006 TABLE 6 Mass Spec Data for Synthesized
Ethylenediamines ##STR00075## R.sub.1NH.sub.2 in [M + 1].sup.+ of
the [M + 1].sup.+ of the products, R.sub.4 = Ph the 1.sup.st
position products, R.sub.4 = H Diamines, 1 Amino alcohols, 13
Tyramine 308 384 258 formed 2-Adamantamine 321 absent 398 absent
272 formed cis-Myrtanyl- 324 400 274 formed amine 3-Amino-1- 246
322 196 absent propanol L-Methioninol 305 absent 382 absent 256
absent Cyclooctylamine 298 374 248 formed (1S,2S)-2-Amino- 337
absent 414 absent 288 absent 1-phenyl-1,3- propandiol 1-Adamantane-
336 412 absent 286 formed methylamine 2,2-Diphenyl- 368 444 318
formed ethylamine 5-Amino-1- 274 350 224 formed pentanol
TABLE-US-00007 TABLE 7 Mass Spec Data for Synthesized
Ethylenediamines, R.sub.4 = H and Me ##STR00076## R.sub.1NH.sub.2
in [M + 1].sup.+ of the [M + 1].sup.+ of the products, R.sub.4 =Me
the 1.sup.st position products, R.sub.4 = H Diamines, 1 Amino
alcohols, 13 Tyramine 278 293 196 absent 2-Adamantamine 293 absent
307 absent 210 low intensity cis-Myrtanyl- 293 309 212 formed amine
3-Amino-1- 217 231 134 absent propanol L-Methioninol 277 absent 291
absent 194 formed Cyclooctylamine 269 269 absent 186 absent
(1S,2S)-2-Amino- 309 low intensity 323 absent 226 formed
1-phenyl-1,3- propandiol 1-Adamantane- 307 321 224 formed
methylamine 2,2-Diphenyl- 339 353 256 formed ethylamine 5-Amino-1-
245 259 162 absent pentanol
TABLE-US-00008 TABLE 8 Mass Spec Data for Synthesized
Ethylenediamines, R.sub.4 = H and Me ##STR00077## R.sub.1NH.sub.2
in [M + 1].sup.+of the [M + 1].sup.+of the products, R.sub.4 =Me
the 1.sup.st position products, R.sub.4 = H Diamines, 1 Amino
alcohols, 13 Tyramine 278 292 absent 196 absent 2-Adamantamine 292
absent 306 absent 210 formed cis-Myrtanyl- 294 308 absent 212
formed amine 3-Amino-1- 216 230 absent 134 absent propanol
L-Methioninol 276 absent 290 absent 194 absent Cyclooctylamine 268
282 absent 186 absent (1S,2S)-2-Amino- 308 322 absent 226 formed
1-phenyl-1,3- propandiol 1-Adamantane- 306 absent 320 absent 224
formed methylamine 2,2-Diphenyl- 338 352 absent 256 formed
ethylamine 5-Amino-1- 244 258 absent 162 absent pentanol
EXAMPLE VI
.sup.1H NMR Spectroscopy
Proton NMR data was recorded on a VARIAN.RTM. Nuclear Magnetic
Resonance Spectrometer (Palto Alto, Calif.) at 500 MHz.
Representative substituted ethylene diamines were purified by HPLC,
and analyzed by proton NMR. A representative proton NMR profiles is
shown in FIG. 16. NMR and MS data for some representative hit
compounds are shown below.
Compound 6.
N.sup.2-(1-Adamantylmethyl)-N-(3,3-diphenylpropyl)propane-1,2-diamine.
55 mg, 36% yield. .sup.1H NMR: .delta. 7.28-7.15 (m, 5H), 3.95 (t,
J=7.9 Hz, M1), 2.94 (br s 4H), 2.71 (dd, J=7.6, 9.8 Hz, 2H), 2.41
(s, 2H), 2.32 (dd, J=7.6, 7.9 Hz, 2H), 2.16 (s), 2.08-1.98 (m, 4H),
1.72 (m, 6H), 1.62 (m, 6H), 1.51 (d, J=2.4 Hz, 3H). Mass spectrum
(ESI) m/z (MH).sup.+ 417.
Compound 7.
N-(3,3-Diphenylpropyl)-N'-(1-adamanthylmethyl)ethane-1,2-diamine.
28 mg, 22% yield. 1H NMR (500 MHz) .delta. 7.30-7.12 (m, 10H); 3.95
(t, J=7.6 Hz, 1H); 2.91 (d, J=1.2 Hz, 4H); 2.70 (dd, J=7.6 and 1.2
Hz, 2H); 2.40 (d, J=1.3 Hz, 2H); 2.32 (q, J=8.0 Hz, 2H); 1.98 (br
d, J=1.7 Hz, 4H); 1.72 (d, J=12.2 Hz, 4H); 1.62 (d, m? J=12.2 Hz,
4H); 1.51 (br s, 6H). Mass spectrum (ESI) m/z (MH).sup.+ 403.6.
Compound 10.
N-(-)-cis-Myrtanyl-N'-(3,3-diphenylpropyl)ethane-1,2-diamine. 14
mg, 11% yield. .sup.1H NMR (500 MHz) .delta. 7.30-7.10 (m, 10H);
3.95 (m, M1); 2.92-2.83 (m, 4H); AB: 2.80 (d, J=7 Hz, 1H); 2.76 (d,
J=8 Hz, 1H); 2.65 (dd, J=9.6 and 7.6 Hz, 2H); 2.42-2.20 (m, 4H),
2.29 (d, J=8 Hz, 2H), 1.90 (m, 8H); 1.42 (m, 1H); 1.19 (m, 2H);
1.17 (s, 3H); 0.95 (s, 3H); 1.00-0.8 (m, 2H). Mass spectrum (ESI)
m/z (MH).sup.+ 391.3.
Compound 14.
N-(3,3-Diphenylpropyl)-N'-exo-(2-norborny)ethane-1,2-diamine. 17
mg, 16% yield. 1H NMR (500 MHz) .delta. 7.30-7.15 (m, 10H); 3.95
(t, J=7.9 Hz, 1H); 2.86 (dd, J=11.5 and 1.5 Hz, 4H); 2.73 (dd,
J=8.0 and 3.3 Hz, 1H); 2.64 (t, J=7.6 Hz, 2H); 2.29 (t, J=7.5 Hz,
2H), 2.31-2.26 (m, 2H) 2.30 1.96 (s, 3H); 1.63 (ddd, J=13.1, 7.9
and 2.5 Hz, 1H); 1.60-1.50 (m, 1H); 1.50-1.43 (m, 2H); 1.30 (dq,
J=4.0 and 13.5 Hz, 1H), (1H, m), 1.20 (dd, J=10.4 and 1.1 Hz, 1H),
1.11 (dd, J=2.0, and 8.5 Hz, 1H), 1.08 (dd, J=2.5, and 8.5 Hz, 1H),
1.10 (dq, J=8.3 and 2.1, 2H). Mass spectrum (ESI) m/z (MH).sup.+
349.1.
Compound 21.
N-(3,3-Diphenylpropyl)-N'-(1S)-(1-ethylcyclohexane)ethane-1,2-diamine.
5 mg, 4% yield. Mass spectrum (ESI) m/z (MH).sup.+ 365.5.
Compound 32.
N-(2,2-Diphenylethyl)-N'-{circle around
(R)}-(+)-bomylethane-1,2-iamine. 58 mg, 48% yield. .sup.1H NMR (500
MHz): .delta. 7.30-7.10 (m, 10H); 4.18 (t, J=6.8 Hz, 1H); 3.34 (d,
J=7.6 Hz, 2H); 3.02 (m, 4H); 2.95-2.90 (m, 1H); 2.15-2.08 (m, 1H);
1.94 (m, 1H); 1.72-1.65 (m, 2H); 1.48-1.30 (m, 2H); 1.27-1.10 (m,
2H); 1.06 (dd, J=13.6 and 4.1 Hz, 1H); 0.82 (s, 3H); 0.81 (s, 3H);
0.78 (s, 3H). Mass spectrum (ESI) m/z (MH).sup.+ 377.2
Compound 34.
N-(2,2-Diphenylethyl)-N'-(1-adamanthylmethyl)ethane-1,2-diamine.
6.8 mg, 6% yield. 1H NMR (500 MHz) .delta. 7.30-7.15 .quadrature.m,
10H); 4.15 (t, J=7.6 Hz, 1H); 3.24 (dd, J=7.9 and 1.2 Hz, 2H); 2.79
(t, J=6.5 Hz, 2H); 2.74 (t, J=6.0 Hz,m, 2H); 1.95 (m, 8H); 1.69 (d,
J=12.5 Hz, 4H); 1.59 (d, J=11.9 Hz, 4H); 1.40 and 1.39 (br s, 3H);
Mass spectrum (ESI) m/z (MH).sup.+ 389.0.
Compound 37.
N-(2,2-Diphenylethyl)-N'-(-)-cis-myrtanylethane-1,2-diamine. 54 mg,
38% yield. .sup.1H NMR: .delta. 7.31-7.18 (m, 10H), 4.13 (t, J=7.6
Hz, 1H), 3.26 (d, J=7.6 Hz, 2H), 2.86 (dd, J=4.3, 8.0 Hz, 4H), 2.76
(dd, J=7.6, 12.2 Hz, 2H), 2.37 (ddd, J=1.8, 9.0, 12.5 Hz, 1H), 2.12
(dq, J=1.8, 7.6 Hz, 1H), 1.98 (br s, 2H), 1.98-1.84 (m, 4H), 1.39
(ddd, J=2.4, 4.0, 6.1 Hz, 1H), 1.18 (s, 3H), 0.95 (s, 3H), 0.91 (d,
J=10.0 Hz, 1H) Mass spectrum (ESI) m/z (MH).sup.+ 377.2.
Compound 38.
N-(-)-cis-Myrtanyl-N'-(2,2-diphenylethyl)propane-1,2-diamine. 39
mg, 30% yield. .sup.1H NMR (500 MHz) .delta. 7.30-7.15 (m, 10H);
4.13 (t, J=8.0 Hz, 1H); AB: 3.28 (d, J=7.5 Hz, 1H); 3.24 (d, J=7.5
Hz, 1H), 3.26 (d, J=6.1 Hz, 2H); 2.96 (m, 1H); 2.88-2.75 (m, 2H);
2.71 (ddd, J=4.5, 9.0, 13.0 Hz, 1H), 2.58 (ddd, J=7.0, 10.0, 14.0
Hz, 1H); 2.35 (m, 1H); 2.21 (m, 1H); 2.00-1.80 (m, 6H); 1.40-1.20
(m, 1H); 1.17 (s, 3H); 0.93 (s, 3H); 0.89 (dd, J=9.7 and 4.2 Hz,
1H). Mass spectrum (ESI) m/z (MH).sup.+ 391.0.
Compound 40.
N-(2,2-Diphenylethyl)-N'-(1R,2R,3R,5S)-(-)-isopinocampheylethane-1,2-diam-
ine. 33 mg, 23% yield. .sup.1H NMR: .delta. 7.31-7.18 (m, 10H),
4.13 (t, J=7.5 Hz, 1H), 3.27 (d, J=8.0 Hz, 2H), 3.14 (dt, J=6.0, 10
Hz, 1H), (4H), 2.36 (qd, J=2.0, 6.0 Hz, 1H), 2.34 (dt, J=2.0, 10
Hz, 1H), 2.07-1.96 (m, 3H), 1.82 (dt, J=2.0, 6.0 Hz, 1H), 1.71
(ddd, J=2.5, 5.5, 13.5 Hz, 1H), 1.22 (s, 3H), 1.09 (d, J=7.0 Hz,
3H), 0.96 (d, J=10.5 Hz, 1H), 0.91 (s, 3H). Mass spectrum (ESI) m/z
(MH).sup.+ 377.3.
Compound 47.
N-(-)-cis-Myrtanyl-N'-(1R,2R,3R,5S)-(-)-isopinocampheylethane-1,2-diamine-
. 42 mg, 33% yield. .sup.1H NMR: .delta. 3.35-3.20 (m, 6H), 2.93
(dd, J=4.6, 2.0 Hz, 2H), 2.45-2.33 (m, 4H), 2.17 (s, 3H), 2.06
(quint, J=7.0 Hz, 1H), 2.0-1.9 (m, 6H), 1.90 (dd, J=2.1, 5.2 Hz,
1H), 1.87 (dt, J=1.8, 4.6 Hz, 1H), 1.51 (ddd, J=4.6, 10.0, 13.0 Hz,
1H), 1.23 (s, 3H), 1.19 (s, 3H), 1.12 (d, J=8 Hz, 3H), 1.03 (d,
J=10.3 Hz, 1H), 0.98 (s, 3H), 0.94 (d, J=9.8 Hz, 1H), 0.94 (s, 3H).
Mass spectrum (ESI) m/z (MH).sup.+ 333.6.
Compound 52.
N-(3,3-Diphenylpropyl)-N'-cyclooctylethane-1,2-diamine. 20 mg, 18%
yield. 1H NMR (500 MHz): .delta. 7.30-7.10 (m, 10H); 3.96 (t, J=7.9
Hz, 1H); 3.00 (m, 1H); 2.90 (dd, J.sub.3=J.sub.2=5.5 Hz, 2H); 2.84
(dd, J.sub.3=J.sub.2=5.0 Hz, 2H); 2.61 (t, J=7.3 Hz, 2H), 2.27 (q,
J=7.6 Hz, 2H); 1.83 (m, 2H); 1.74 (m, 2H); 1.65-1.40 (m, 10H).
Compound 55.
N-(1-Adamantylmethyl)-N'-cyclooctylethane-1,2-diamine. 6.7 mg, 6%
yield. 1H NMR (500 MHz): .delta. 3.08-3.02 (m, 1H), 3.02-2.98 (m,
2H); 2.97-2.92 (m, 2H); 2.36 (s, 2H); 1.98 (m, 2H); 1.93-1.86 (m,
2H); 1.80-1.50 (m, 19H).
Compound 57.
N-(-)-cis-Myrtanyl-N'-(cyclooctyl)ethane-1,2-diamine. 18 mg, 18%
yield. 1H NMR (500 MHz) .delta. 3.05-2.95 (m, 4H); AB: 2.76 (d,
J=7.5 Hz, 1H), 2.23 (d, J=8.0 Hz, 1H); 2.76 (dd, J=11.6 and 7.3 Hz,
1H); 2.73 (dd, J=11.9 and 8.2 Hz, 1H); 2.40-2.34 (m, 1H); 2.28
(quintet, J=8.0 Hz, 1H); 1.97 (s, 3H); 2.00-1.84 (m, 6H); 1.80-1.70
(m, 2H); 1.68-1.38 (m, 1H); 1.18 (s, 3H); 0.97 (s, 3H); 0.92 (d,
J=9.8 Hz, 1H). Mass spectrum (ESI) m/z (MH).sup.+ 307.5.
Compound 58.
N-(2-Adamantyl)-N'-cyclooctylethane-1,2-diamine. 25 mg, 23% yield.
.sup.1H NMR: .delta. 3.06 (m, 1H), 3.00 (t, J=6.1 Hz, 2H), 2.93 (t,
J=5,5 Hz, 2H), 2.83 (br s, 1H), 1.96 (s, 3H), 1.92-1.80 (m, 10H),
1.80-1.50 (m, 20H). Mass spectrum (ESI) m/z (MH).sup.+ 305.1.
Compound 59.
N-(Cyclooctyl)-N'-(1R,2R,3R,5S)-(-)-isopinocampheylethane-1,2-diamine.
15 mg, 14% yield. 1H NMR (400 MHz): .delta. 3.47 (dt, J=6.0, 10.0
Hz, 1H), 3.40-3.28 (m, 7H), 2.44 (tq, J=2.0, 10.0 Hz, 1H), 2.36
(dtd, J=2.0, 6.0, 10.0 Hz, 1H), 2.09 (dq, J=2.0, 7.2 Hz, 1H),
2.00-1.90 (m, 3H), 1.88-1.78 (m, 2H), 1.78-1.63 (m, 4H), 1.65-1.30
(m, 8H), 1.18 (d, J=6.0 Hz, 3H), 1.16 (s, 3H), 1.17 (d, J=7.2 Hz,
1H), 0.90 (s, 3H). Mass spectrum (ESI) m/z (MH).sup.+ 307.4.
Compound 62.
N-(-)-cis-Myrtanyl-N'-(1S)-(1-ethylcyclohexane)ethane-1,2-diamine.
48 mg, 46% yield. 1H NMR (500 MHz): .delta. 3.06-3.00 (m, 1H);
2.98-2.95 (m, 2H); 2.92-2.84 (m, 1H); 2.79 (dd, J=11.9 and 7.0 Hz,
1H); 2.75 (dd, J=11.9 and 7.9 Hz, 1H); 2.73 (m, 1H); 2.39 (m, 1H);
2.28 (quintet, J=8.5 Hz, 1H); 2.00-1.86 (m, 6H); 1.82-1.76 (m, 2H);
1.68 (m, 2H); 1.54-1.42 (m, 2H); 1.32-1.10 (m, 6H); 1.19 (s, 3H);
1.13 (d, J=6.7 Hz, 3H); 1.07 (dd, J=12 and 3 Hz, 2H); 1.02 (dd,
J=12 and 3 Hz, 2H); 0.98 (s, 3H); 0.93 (d, J=9.7 Hz, 1H). Mass
spectrum (ESI) m/z (MH).sup.+ 306.9.
Compound 65.
N-trans-(2-phenylcyclopropyl)-N'-(1-adamanthyl)ethane-1,2-diamine.
18 mg, 16% yield. Mass spectrum (ESI) m/z (MH).sup.+ 311.3.
Compound 66.
N-(3,3-Diphenylpropyl)-N'-(1R,2R,3R,5S-(-)-isopinocampheylethane-1,2-diam-
ine. 2 mg, 2% yield. 1H NMR (500 MHz) .delta. 7.26 (m, 10H); 3.96
(t, J=7.6 Hz, 1H); 3.09 (m, 1H); 2.92 (m, 1H); 2.84 (m, 2H); 2.62
(m, 2H); 2.35 (m, 4H); 1.97 (s, 3H); 1.82 (m, 1H); 1.68 (m, 1H);
1.21 (s, 3H); 1.12 (d, J=7.3 Hz; 3H); 1.01 (m, 1H); 0.92 (s, 3H).
Mass spectrum (ESI) m/z (MH).sup.+ 391.4.
Compound 73.
N-(2-Adamantyl)-N'-[2-(2-methoxyphenyl)ethyl]ethane-1,2-diamine. 21
mg, 19% yield. .sup.1H NMR: .delta. 7.22 (dd, J=8.2, 7.3 Hz, 1H),
7.14 (d, J=7.3 Hz, 1H), 6.89 (d, J=7.1, Hz, 1H), 6.87 (d, J=8.2,
Hz, 1H), 3.81 (s, 3H), 3.06 (t, J=7.1 Hz, 2H), 3.06 (m, 2H), 3.01
(m, 2H), 2.93 (t, J=7.1, 2H), 1.95 (br s, 2H), 1.90-1.80 (m, 7H),
1.78-1.66 (m, 6H), 1.59 (d, J=2.5 Hz, 2H). Mass spectrum (ESI) m/z
(MH).sup.+ 329.4.
Compound 78.
N-2-Adamantyl-N'-2,3-dihydro-1H-inden-2-yl-ethane-1,2-diamine. 4.3
mg, 3% yield. .sup.1H NMR: .delta. 7.20 (dd, J=4.9, 8.5 Hz, 2H),
7.14 (dd, J=5.5, 2.1 Hz, 2H), 3.71 (quint, J=6.1 Hz, 2H), 3.19 (dd,
J=5.8, 15.9 Hz, 2H), 3.13 (br.s, 1H), 3.05 (m, 4H), 2.86 (dd,
J=4.8, 15.8 Hz, 2H), 2.08 (m, 2H), 2.00 (m, 6H), 1.96-1.88 (m, 4H),
1.88-1.80 (m, 3H), 1.74 (m, 4H), 1.68-1.60 (m, 2H). Mass spectrum
(ESI) m/z (MH).sup.+ 303.4.
Compound 109.
N-Geranyl-N'-(2-adamanthyl)ethane-1,2-diamine. 27 mg, 24% yield. 1H
NMR (400 MHz): .delta. 5.40 (t, J=7.2 Hz, 1H), 4.78 (br s, 2H),
3.64 (d, J=7.6 Hz, 2H), 3.34 (m, 2H), 2.07 (m, 2H), 2.08-1.95 (m,
4H), 1.95-1.85 (m, 4H), 1.82 (m, 2H), 1.88-1.70 (m, 4H), 1.70-1.62
(m, 3H), 1.67 (s, 3H), 1.56 (s, 3H), 1.50 (s, 3H). Mass spectrum
(ESI) m/z (MH).sup.+ 307.4.
Compound 111.
N-Geranyl-N'-(2-ethylpiperidine)ethane-1,2-diamine. 44 mg, 42%
yield. 1H NMR (500 MHz): .delta. 5.22 (t, J=6.1 Hz, 1H); 5.04 (m,
1H), 3.52 (d, J=7.3 Hz, 2H); 3.05-2.85 (m, 4H); 2.66 (m, 1H); 2.44
(m, 2H); 2.08 (m, 4H); 1.80-1.50 (m, 2H); 1.70(s, 3H); 1.65 (s,
3H); 1.58 (s, 3H); 1.50-1.35 (m, 2H), 0.89 (t, J=7.3, 3H). Mass
spectrum (ESI) m/z (MH).sup.+ 293.4.
Compound 116.
N-Geranyl-N'-allyl-N'-(cyclopentyl)ethane-1,2-diamine. 45 mg, 42%
yield. .sup.1H NMR: .delta. 5.86 (ddd, J=10.0, 16.1, 6.7 Hz, 1H),
5.28 (d, J=15.9 Hz, 1H), 5.25 (d, J=8.7 Hz, 1H), 5.23 (t, J=7.3 Hz,
1H), 5.30 (m, 1H), 3.59 (d, J=7.3 Hz, 2H), 3.28 (br d, J=6.4 Hz,
2H), 3.16 (quintet, J=8.2 Hz, 1H), 3.02 (m, 2H), 2.95-2.86 (m, 2H),
1.88-1.80 (m, 4H), 1.70 (s, 3H), 1.74-1.66 (m, 3H), 1.65 (s, 3H),
1.58 (s, 3H), 1.56-1.50 (2H), 1.50-1.40 (m, 2H). Mass spectrum
(ESI) m/z (MH).sup.+ 305.3.
Compound 117.
N-Geranyl-N'-diphenylmethylethane-1,2-diamine. 24 mg, 20% yield. 1H
NMR (500 MHz): .delta. 7.40 (d, J=7.2 Hz, 4H); 7.29 (t, J=7.3 Hz,
4H); 7.21 (t, J=7.0 Hz, 2H); 5.15 (t, J=7.5, 1H); 5.01 (m, 1H);
4.89 (br s, 1H); 3.42 (d, J=7.0 Hz, 2H); 3.00-2.78 2.93 (m, 4H);
2.20-2.00 2.17 (m, 4H); 1.63 (s, 3H); 1.59 (s, 3H); 1.56 (s, 3H).
Mass spectrum (ESI) m/z (MH).sup.+ 363.3.
Compound 125.
N,N'-bis-(-)-cis-Myrtanylpropane-1,2-diamine. 82 mg, 70% yield. 1H
NMR (500 MHz): .delta. 3.62 (m, 1H); 3.18 (dd, J=13.7 and 3.7 Hz,
1H); 3.05 (dt, J=11.5 and 7.5 Hz, 1H); 3.06-2.92 (m, 2H); 2.86 (dt,
J=12.2 and 7.3 Hz, 1H); 2.40 (m, 4H); 2.06-1.84 (m, 10H); 1.56-1.46
(m, 2H); 1.37 and 1.36 (two d, J=6.7 and J=7.0 Hz, 3H); 1.20 (s,
3H); 1.19 (m, 3H), 0.99 and 0.98 (two s, 3H) Hz, H); 0.97 (s, 3H);
0.94 (two d, J=10.1 Hz, 2H). Mass spectrum (ESI) m/z (MH).sup.+
346.9.
Compound 151.
N-[2-(2-Methoxy)phenylethyl]-N'-(1R,2R,3R,5S)-(-)-isopinocampheyl-ethane--
1,2-diamine. 67 mg, 60% yield. 1H NMR (500 MHz): .delta. 7.23 (t,
J=5.8 Hz, 1H); 7.13 (dd, J=5.8 and 1.8 Hz, 1H); 6.88 (m, 2H); 3.81
(s, 3H); 3.13 (m, 1H); 3.1-3.0 (m, 3H); 3.01 (t, J=7.0 Hz, 2H);
2.89 (t, J=7.0 Hz, 2H); 2.42-2.35 (m, 2H); 2.00 (m, 3H); 1.82 (dt,
J=6.0 and 2.0 Hz, 1H); 1.72 (ddd, J=2.5, 5.5, 13.5 Hz, 1H); 1.22
(s, 3H) 1.13 (d, J=7.3 Hz, 3H). 0.99 (d, J=10.1 Hz, 1H); 0.93 (s,
3H). Mass spectrum (ESI) m/z (MH).sup.+ 331.5.
N-2-(2-Methoxyphenyl)ethyl-N'-allyl-N'-cyclopentyl-ethane-1,2-diamine.
8 mg, 7% yield. .sup.1H NMR: .delta. 7.26 (dd, J=7.3, 8.5, 1H),
7.18 (d, J=7.2 Hz, 1H), 6.91 (m, 2H), 5.61 ddd, (J=6.7, 17.0, 9.4
Hz, 1H), 5.13 (d, J=15.3 Hz, 1H), 5.10 (d, J=9.2 Hz, 1H), 3.83 (s,
3H), 3.13 (dd, J=7.0, 6.7 Hz, 2H), 3.10 (d, J=6.7 Hz, 1H), 3.00 (d,
J=7.3 Hz, 1H), 3.05-2.90 (m, 2H), 2.97 (dd, J=8.2, 6.1 Hz, 2H),
2.75 (t, J=6.1 Hz, 2H), 1.73 (m, 2H), 1.62 (m, 2H), 1.50 (m, 2H),
1.22 (m, 2H). Mass spectrum (ESI) m/z (MH).sup.+ 311.4.
N.sup.2-(3-Phenylpropyl)-N'-[2-(4-fluorophenyl)ethyl]-1-phenylethane-1,2--
diamine. 23 mg, 19% yield. .sup.1H NMR: .delta. 7.35 (d, J=7.6 Hz,
2H), 7.34 (quart, J=7. Hz, 1H), 7.26 (d, J=6.4 Hz, 3H), 7.23 (d,
J=7.6 Hz, 2H), 7.17 (dd, J=7.3, 6.4 Hz, 1H), 7.12 (d, J=7.0 Hz,
2H), 3.21 (m, 1H), 3.03 (ddd, J=4.2, 8.0, 12.8 Hz, 4H), 2.86 (t,
J=8.0 Hz, 2H), 2.85-2.79 (m, J=12. Hz, 2H), 2.74-2.64 (m, 4H), 2.61
(t, J=7.7 Hz, 2H), 1.96 (quint, J-=7.6 Hz, 2H). Mass spectrum (ESI)
m/z (MH).sup.+ 377.3.
EXAMPLE VII
M. Tuberculosis Rv0341p Lucs Drug Response
Substituted ethylene diamines, as described herein, were tested on
Mycobacterium tuberculosis using high-throughout screening assay
with recombinant mycobacterial containing promoter fusion of
luciferase to Rv0341 EMB-inducible promoter. This assay quickly and
reliably identifies antimycobacterial activity in compound mixtures
and/or in individual compounds. In this assay, bioluminescence
increases when the mycobacteria is tested against an active
compound, or an active compound mixture. During this assay, a
theoretical yield of 100% was assumed for every unpurified
substituted ethylene diamine, and the activity of each sample was
compared to commercially available ethambutol (99.0% purity).
Results were reported in LCPS, and % Max. LCPS based on the
activity of EMB at 3.1 .mu.M.
The substituted ethylene diamines were analyzed according to the
following procedure. The diamines were dried in a speed vacuum to
an approximate concentration of 6.3 mmoles per well. Each diamine,
or diamine mixture, was then resuspended or dissolved in 200 .mu.l
of methanol for a concentration of 31.5 mM diamine(s). The
diamine(s) solution was diluted to a concentration of 200 .mu.M in
7H9 broth medium (a 1:15.75 dilution of the 31.5 mM stock, followed
by a 1:10 dilution; each dilution in 7H9 broth medium). Next, 50
.mu.l of the diluted diamine(s) solution was added to the first
well of a row of twelve in an opaque, 96-well plate. The 7H9 broth
medium, 25 .mu.l, was added to each of the remaining wells (#2-12)
in the row. The diamine(s) solution in "well one" was serially
diluted by transferring 25 .mu.l from "well one" to "well two", and
repeating a 25 .mu.l transfer from "well two" to "well three", and
so on, on through "well eleven". In "well eleven", the extra 25
.mu.l of solution was discarded. "Well twelve" was used as a growth
control to assess background activity of the reporter strain. The
plate was then covered and incubated at 37.degree. C. for 24 hours.
Immediately prior to analysis, the following substrates were
prepared: a buffer solution containing 50 mM HEPES at pH 7.0 and
0.4% Triton X-100. Then, 0.25 ml of 1M DTT, and 14 Itl of 10 mg/ml
luciferin in DMSO were added to 5 ml of the buffer solution. This
final solution (50 .mu.l) was added to each of the twelve wells,
immediately after the incubation period had run. The luminescence
from each well was measured 20 minutes after the luciferin
substrate was added, using a TOPCOUNT.RTM. (Downers, Grove, Ill.)
NXT luminometer (55/well).
FIGS. 6-8 show typical assay data for the luciferase reporter
strain containing an Rv0341 EMB-inducible promoter with serial
dilution of 12 wells (1 row) of a 96-well library plate. FIG. 10
shows the number of substituted ethylene diamines with at least 10%
luciferase activity, based on the activity of ethambutol at 3.1
.mu.M.
FIG. 6 represents typical assay data in the luciferase reporter
strain containing an Rv0341 EMB-inducible promoter. The data
represents values obtained from the HTS Luc assay for compound
mixtures of one row (row D) in the 96-well library. Row D was
subject to several serial dilutions. The effectiveness of the
compound mixture in the assay was measured by the intensity of
luminescence, and compared to ethambutol (100% intensity, 99%
purity) at 3.1 .mu.M. Each curve in FIG. 6 represents one well, or
ten compounds. Results are reported in percent maximum Luminescence
Count per Second (% Max. LCPS). During the screening, a theoretical
100% chemical yield was assumed for every unpurified compound.
Concentrations are given for a single compound. Based on this
initial screening, 300+ compound mixtures showed anti-TB
activity.
EXAMPLE VIII
Representative MIC Experiment
The Minimum Inhibition Concentration (MIC) is the concentration of
the growth inhibitor, here a substituted ethylene diamine, at which
there is no multiplication of seeded cells. A microdilution method
was used to determine the MIC of the substituted ethylene diamines,
capable of inhibiting the growth of Mycobacterium tuberculosis in
vitro. In a representative MIC experiment, bacteria, the H37Rv
strain of Mycobacterium tuberculosis (M.tb), was cultivated in 7H9
medium to a density of 0.2 OD (optical density) at 600 nm. The
bacterial culture was then diluted 1:100 in 7H9 broth medium. Stock
solutions of isoniazid and ethambutol were each prepared at 32
.mu.g/ml in 7H9 medium. A 3.2 mg/ml solution of isonizid and
ethambutol were each prepared in water. The solutions were then
filtered, and diluted 1:100 in 7H9 medium. Each drug, purchased
from Sigma, was "laboratory use only" grade. A 10 mM solution of
each substituted ethylene diamine was prepared in methanol. Next,
100 .mu.l of the 7H9 medium was added to each well in a 96-well
plate (rows (A through H) x columns (1 through 12)). To the first
wells in rows C through H was added an additional 80 .mu.l of the
7H9 medium. The isoniazid solution, 100 .mu.l, was added to well
A1, and the ethambutol solution, 100 .mu.l, was added to well B1.
Six substituted ethylene diamines, 20 .mu.l each, were added to
wells C1 through H1 (column 1), respectively. A serial dilution of
each substituted ethylene diamine and the isoniazid and ethambutol
controls, was performed across each row. For example, a serial
dilution across row C.sub.1-C.sub.12 was done by mixing and
transferring 100 .mu.l of the previous well to the next consecutive
well. In each well in "column 12," 100 .mu.l of the final dilution
was discarded. Next, 100 Ill of the diluted H37Rv strain of M.tb
was added to each well. The 96-well plate was then covered and
incubated at 37.degree. C. for 10 days. The plate was read for
bacterial growth, or non-growth, using an inverted plate reader.
The MIC was determined to be the lowest concentration of
substituted ethylene diamine that inhibited visible growth of the
M.tb.
A representative plate layout, listing concentration in each well,
is shown in Table 9. Table 10 lists MIC and LD50 data for selected
compounds. The LD50 is the concentration of the substituted
ethylene diamine at which 50% of the cells (R.sub.37Rv strain of
M.tb) are killed. Table 11 lists MIC data for purified substituted
ethylene diamines in comparison to ethambutol (EMB). FIG. 9 shows
the number of substituted ethylene diamine compounds with MIC
activity at various concentration levels.
TABLE-US-00009 TABLE 9 Concentration in Each Well (.mu.M) Based on
Columns 1-12 DRUG Isoniazid 58.25 29.13 14.56 7.28 3.64 1.82 0.91
0.45 0.23 0.11 0.06 0.03 Ethambutol 28.75 14.38 7.19 3.60 1.80 0.90
0.45 0.22 0.11 0.06 0.03 0.01 Subst. 500 250 125 62.5 31.25 15.63
7.81 3.91 1.96 0.98 0.49 0.24 Ethylene Diamine
TABLE-US-00010 TABLE 10 Selectivity Index for Selected Compounds
MIC LD50 LD50 Cmpd (uM) (uM) MW MIC (ug/ml) (ug/ml) SI 6 7.813 20
536 4.187768 10.72 2.559836 34 7.813 32 508 3.969004 16.256
4.095738 37 15.625 32 496 7.75 15.872 2.048 47 15.625 25 452 7.0625
11.3 1.6 57 15.625 18 426 6.65625 7.668 1.152 59 15.625 32 426
6.65625 13.632 2.048 65 15.625 60 430 6.71875 25.8 3.84 109 1.953
32 450 0.87885 14.4 16.38505 111 7.813 44 412 3.218956 18.128
5.63164 151 7.813 41 450 3.51585 18.45 5.247664
The above procedure was also used to examine batched compounds (10
compounds per well). Synthesized batches of substituted ethylene
diamines were dried in speed vacuum and then resuspended in DMSO or
sterile water to a concentration of 2.5 mg/ml.
TABLE-US-00011 TABLE 11 MIC Data for Purified Samples Plate set-up
INH 58.25 29.125 14.56 7.28 3.64 1.82 0.91 0.45 0.23 EMB 28.75
14.375 7.1875 3.594 1.797 0.898 0.449 0.2245 0.1125 CMPD 500 250
125 62.5 31.25 15.625 7.813 3.9063 1.953 Avg INH MIC (uM) Avg INH
MIC (uM) 0.91 0.91 Avg EMB MIC (uM) Avg EMB MIC (uM) Avg EMB Avg
EMB 7.1875 8.37 7.25 7.25 BACTEC (EMB: Cmpd MIC (uM) 2.5 UG/ML) 1
250 250 125 125 2 250 250 250 250 3 31.25 62.5 15.6 15.6 4 125 62.5
62.5 62.5 5 >500 500 500 500 6 7.813* 7.813 3.9 3.9 7 15.625*
7.813 3.9 3.9 8 125 125 31.25 31.25 10 7.813* 15.625 7.8 7.8 11
31.25 contaminated 3.9 3.9 13 31.25 31.25 15.6 15.6 15 14 15.625''
15.625 7.8. 7.8 15 >500 >500 250 500 17 62.5 62.5 15.6 15.6
21 15.625* 31.25 7.8 7.8 22 31.25 31.25 7.8 15.6 23 31.25 31.25
15.6 15.6 24 125 125 31.25 31.25 27 125 62.5 15.6 31.25 28 125 62.5
31.25 31.25 29 62.5 62.5 31.25 62.5 31 31.25 61.25 15.6 15.6 32
15.625* 15.625 7.8 7.8 33 62.5 62.5 31.25 31.25 34 7.813* 7.813 3.9
3.9 35 62.5 62.5 15.6 31.25 36 31.25 62.5 15.6 15.6 37 15.625*
15.625 3.9 7.8 1.25 38 7.813 7.813 3:9 7.8 40 15.625* 15.625 7.8
7.8 41 31.25 15.625 15.6 15.6 42 31.25 31.25 1.95 3.9 43 31.25
31.25 3.9 7.8 12.5 47 15.625* 15.625 1.95 7.8 5 51 31.25 250 31.25
31.25 52 15.625* 15.625 3:9 3.9 53 31.25 31.25 31.25 31.25 54 31.25
31.25 15.6 31.25 55 15.625* 15.625 15.6 15.6 25 56 500 >500 500
500 57 15.625* 7.813 7.8 7.8 58 15.625* 15.625 7.8 7.8 5 59 15.625*
31.25 15.6 15.6 12.5 61 62.5 62.5 31.25 31.25 62 15.625* 31.25 15.6
31.25 63 62.5 62.5 31.25 62.5 64 31.25 31.25 31.25 31.25 65 15.625*
31.25 31.25 31.25 66 15.625* 15.625 7.8 7.8 68 500 500 500 500 71
62.5 62.5 31.25 31.25 73 62.5 15.6 15.6 76 62.5 62.5 31.25 31.25 77
31.25 31.25 15.6 15.6 78 15.625* 31.25 15.6 15.6 79 31.25 31.25
15.6 15.6 103 31.25 31.25 62.5 62.5 107 500 500 250 250 109 1.953*
1.953 1.95 1.95 0.63 111 7.813* 7.813 7.8 7.8 5 116 15.625* 15.625
7.8 15.6 12.5 117 7.813* 15.625 7.8 7.8 118 31.25 62.5 31.25 no
data 119 125 62.5 cont no data contam 125 15.625* 15.625 cont no
data 6.25 134 >500 >500 500 no data 151 15.625* 7.813 cont no
data 6.25 164 62.5 125 cont no data 165 62.5 62.5 15.6 15.6
EXAMPLE IX
Secondary Screening and Evaluation of Substituted Ethylene Diamines
Against Drug Resistant Patient Isolates
Secondary screening was performed on some of the substituted
ethylene ne compounds to examine their activity against three
clinically resistant MDR t isolates. MDR Strain TN576 is classified
as a Wl strain (STP.sup.R, INH.sup.R, RIF.sup.R, ETH.sup.R,
KAN.sup.R, CAP.sup.R) strain TN587 is classified as a W strain
(STP.sup.R, INH.sup.R, EMB.sup.R, KAN.sup.R), and the third strain
TN3086 is classified as a Wl strain (STP.sup.R, RIF.sup.R,
EMB.sup.R, KAN.sup.R). Each MDR strain is highly resistant to
ethambutol with values exceeding 12.5-25 .mu.M. The MICs for the
following substituted ethylene nes, MP 116, MP 117, RL 241,
compounds #59 and #109, were determined for all patient
isolates.
##STR00078##
The results from this study are shown in Tables 12-13. Table 14
characterizes each MDR strain according to its resistance.
TABLE-US-00012 TABLE 12 Screening of Substituted Ethylene Diamines
Against Drug Resistant Patient Isolates - (MIC values in ug/ml) WT
576 587 3806 EMB 3.12 12.5-25 12.5-25 12.5-25 (or 11.1 uM) MP 116
6.25 3.15 6.25 3.15 MP 117 6.25 3.15 3.15 3.15 RL 241 1.5 1.5 1.5
1.5 (or 3.34 uM) WT = wild type of M.tb EMB as 2HCl salt RL241 as
2HCl salt
TABLE-US-00013 TABLE 13 Screening of Substituted Ethylene Diamines
Against Drug Resistant Patient Isolates - (MIC values in ug/ml) WT
576 587 3806 EMB 1.6-1.8 50 50 50 Cmpd#59 0.05 (or 0.13 uM) 0.1
0.05 0.05 Cmpd#109 0.10 (or 0.18 uM) 0.2 0.2 0.1 Cmpd#59 as a 2HCl
salt Cmpd#109 as a 2CF.sub.3COOH salt
TABLE-US-00014 TABLE 14 Drug Resistance of Each MDR Strain Strain
STP STP 2 INH 1 INH 2 Rif Emb Eth Kan Cip Cap Cyc 576 R R R R R R R
R S R S W1 587 W R R R R R R S R S S S 3806 R R R R S R W1 R =
resistant S = susceptible STP = Streptomycin INH = Isoniazid Rif =
Rifampicin Emb = Ethambutol Eth = Ethionamide Kan = Kanamycin Cip =
Ciprofloxacin Cap = Capreomycin Cyc = Cycloserine
EXAMPLE X
In Vivo Animal Studies
Animal models were used in the final stages of the drug discovery
cycle to assess the anti-microbial efficacy of some substituted
ethylanediamine compounds in a representative system of human
disease state. The in vivo testing approach involves the
inoculation of four-six week old C57BL/6 mice via aerosol,
containing approximately 200 colony forming units of M.
tuberculosis H37Rv.
A. CFU Lung Study
Mice aerosolized with M. tuberculosis H37Rv were examined for 10 to
12 weeks following inoculation. Drugs (substituted ethylene
diamines) were administered via the esophageal cannula (gavage) 7
days/week, starting at either 14 or 21 days post infection.
Bacterial load in the lungs of five mice per group were determined
at approximately one-week intervals by viable colony counts. The
drugs tested were directly compared to the front line
anti-tuberculosis drug isoniazid, and to the second line drug,
ethambutol. Isoniazid and ethambutol were tested at 25 mg/kg and
100 mg/kg, respectively. The substituted ethylene diamines,
compound 37, compound 59 and compound 109, were each tested at 1
mg/kg and 10 mg/kg. FIGS. 17 to 19 represent data from three,
independent CFU Lung studies. In each study, the number of colony
forming units (CFU) that were recoverable and cultivatable, were
determined during various time intervals (days).
B. Lesion Study
The ability of compound 59 and compound 109 to prevent the
development of gross pathology due to bacterial burden was
determined in conjunction with the CFU/Lung Study. The gross
pathology was determined by visible quantitation of lesions on the
surface of the lungs. Quantitation by inspection is a good
surrogate for CFU determination, and directly correlates to the
bacterial burden, as determined by the actual colony forming units.
The lesions are first visibly examined, and then the lungs are
processed and plated for CFU quantification. The lesion study
demonstrates the ability of the drug to prevent the development of
the disease pathology. FIG. 20 represents data from a lesion study.
The corresponding CFU results are shown in FIG. 19.
C. Toxicity Study
Toxicity was assessed using a dose escalation study. This study was
performed with ten C57BL/6 mice per candidate. Every two days, the
mice were administered an increased concentration of the drug, and
monitored for detrimental effects. The administration scheme was
50, 100, 200, 400, 600, 800 and 1000 mg/kg. The maximum limit of
1000 kg/mg was based on the goal of dose escalation, and the
solubility of the drugs in the delivery vehicle. Compound 37 was
toxic in mice at 100 kg/mg. Compound 59 and compound 109 were
tolerated in mice at 1000 mg/kg and 800 mg/kg, respectively.
It should be understood that the foregoing relates only to
preferred embodiments of the present invention, and that numerous
modifications, or alterations, may be made therein without
departing from the spirit and scope of the invention. The entire
text of each reference mentioned herein is hereby incorporated, in
its entirety, by reference.
EXAMPLE XI
In Vitro Toxicity and Selectivity Indexes for Hit Compounds
Twenty six compounds (including 37, 59 and 109) were tested in an
in vitro model of toxicity using monkey kidney cells (Vero) and
human cervical cancer cells (HeLa) using methods well known to
those skilled in the art. The data from this toxicity testing and
the MIC data were used to calculate a selectivity index (SI), the
ratio of IC50:MIC (Table 15). Selectivity Indexes were ranging from
1.76 to 16.67. Compound 109 has the best selectivity index.
TABLE-US-00015 TABLE 15 In vitro data for representative compounds.
Compound MIC (.mu.M) Vero IC50 (.mu.M) SI (IC50:MIC) 66 15.6 28
1.76 40 15.6 25 1.88 41 3.13 19 2.05 59 15.6 36 2.30 55 15.6 34
2.32 57 11.7 22 2.40 37 7.8 32 4.10 38 6.25 33 5.28 111 7.81 45
5.76 73 12.5 81 6.48 58 12.5 82 6.56 78 15.6 130 8.33 109 1.56 26
16.67
EXAMPLE XII
In Vivo Efficacy of Ethambutol Analogues
Compounds 58, 59, 73, 109, and 111 were selected for in vivo
efficacy studies in a mouse model of TB. Compounds 58 and 59 share
the same cyclooctyl fragment in their molecules; compounds 58, 73,
and 109 share adamantly moiety, and 109 and 111--the geranyl
fragment (FIG. 22).
In these studies, 8-week old inbred female mice C57BL/6 were
intravenously infected with M. tuberculosis. 3 weeks following
infection drug treatment was initiated (detailed protocol is
provided). The drugs were administered orally by gavage. Mice were
sacrificed at three timepoints (15, 30, and 45 days post
infection), and CFUs in spleen and lungs were determined (FIGS. 23
and 24). These studies demonstrated that compound 109 had activity
at doses 1 and 10 mg/kg equal to that of ethambutol at 100
mg/kg.
Materials and Methods
Mice. Female C57BL/6 mice of 8 weeks old were purchased from
Charles River (Raleigh, N.C.), housed in BSL-2 facility of BIOCAL,
Inc. (Rockville, Md.), and were allowed to acclimate at least 4
days prior infection.
Mycobacteria. An example of frozen and thawed of M. tuberculosis
H37Rv Pasteur was added to 5 ml 7H10 broth medium, with 0.5% BSA
and 0.05% Tween 80, incubated 1 week at 37.degree. C., and then 1
ml was added into 25 ml medium (2-d passage during 2 weeks).
Culture was washed twice and resuspended in PBS with 0.5% BSA and
0.05% Tween 80, aliquoted and frozen at -80.degree. C. To
determined CFU of the culture aliquot was thawed, and 10-fold
dilutions will be plated on agar 7H9 and CFU count will be
calculated 20 days later.
Infection: Frozen sample of culture was thawed, and diluted for
concentration about 10.sup.6 CFU/ml. Mice were infected with M.
tuberculosis H37Rv intravenously through lateral tail vein in
corresponded dose in 0.2 ml of PBS.
Antimicrobial agents. INH, EMB, Ethambutol analogues.
Protocol of drug treatment: Treatment of mice with compounds was
initiated 20 days following infection. Compounds were dissolved in
10% ethanol in water and administered by gavage (0.2 ml per mouse).
Therapy was given 5 days per week and continued for four or six
weeks. Two, four and six weeks following chemotherapy start mice (6
mice per group) were sacrificed, lungs and spleens were removed and
homogenized in sterile in 2 ml PBS with 0.05% Tween-80. Homogenates
were plated in serial dilutions on 7H10 agar dishes, and incubated
at 37.degree. C. CFU counts were calculated three weeks later.
Statistic analysis. To analyze results of CFUs in organs ANOVA test
was performed; the significance of the differences was estimated by
Student's test, p<0.05 was considered statistically
significant.
Results
In vivo activities of new compounds. The activities of these
compounds are presented in FIGS. 21-24. In the experiment presented
in FIGS. 21 (spleen) and 22 (lung) mice were infected with
5.times.10.sup.5 CFU M. tuberculosis H37Rv and chemotherapy was
started 20 days following infection. Mice were treated with INH (25
mg/kg), EMB (100 mg/kg), compounds 73 and 109 (both 1 mg/kg and 10
mg/kg). The results indicate that in the spleen, compounds 73 and
109 have activities equal to that of EMB at 100 mg/kg (FIG. 21). In
spleen there are no statistical differences between activities of
these compounds at 1 mg/kg or 10 mg/kg. In the lung, compound 109
at concentration 10 mg/kg after 4 and 6 weeks was more effective
than EMB at 100 mg/kg. In the lung, statistically sufficient
difference was shown for compound 109 at concentrations 1 mg/kg and
10 mg/kg (FIG. 22). INH was the most active drug in both spleen and
lung.
Compounds 73 and 109 were also tested in shorter model with using
higher dose of infection (FIGS. 23 and 24). Mice were infected with
5.times.10.sup.6 CFU M. tuberculosis H37Rv and chemotherapy was
started 15 days following infection. Mice were treated with INH (25
mg/kg), EMB (100 mg/kg), compounds 109 (0.1 mg/kg, 10 mg/kg, and 25
mg/kg), 58, 73 and 111 (all 25 mg/kg). Mice were treated for 4
weeks. In both the spleen and lung, compound 109 at concentrations
10 mg/kg and 25 mg/kg had activity equal to that of EMB at 100
mg/kg, and at concentration 0.1 mg/kg minimal but sufficient
difference with untreated control appeared after 4 weeks of therapy
(FIGS. 23 and 24). Statistically sufficient difference between
compounds 73 (25 mg/kg) and 109 (25 mg/kg) was detected. In the
lung significant difference between activities of these compounds
was not detected. Compounds 58 and 111 are active in vivo in both
spleen and lung; however, compounds 73 and 109 are preferable. The
results of these experiments indicate that compounds 73 and 109 in
low concentration show activity equal that of EMB at 100 mg/kg, and
in some cases compound 109 shows higher activity.
Testing of compounds 111 and 59 was performed in B6 mice infected
with 5.times.10.sup.5 CFU M. tuberculosis H37Rv and beginning
chemotherapy 20 days following infection (FIGS. 25 and 26). Both
compounds showed anti-tuberculosis activity at concentration 10
mg/kg comparable to that of EMB at 100 mg/kg.
In all experiments, INH showed higher activity than EMB and other
compounds decreasing load of bacteria in organs on 2-3 logs during
4-6 weeks of chemotherapy; new compounds similar to EMB (100 mg/kg)
decreased load of bacteria on 1.0-2.0 logs. Among studied compounds
73 and 109 are the most preferable, because the highest capacity to
decrease mycobacteria in organs and its parameters of toxicity and
pharmacology kinetics.
EXAMPLE XIII
In Vivo Toxicity
Preliminary dose acceleration studies in mice have indicated that
compound 109 can be well tolerated at doses up to 800 mg/kg and
compound 59 up to 1000 mg/kg. Compound 37 was fatal at doses 100
mg/kg (Clif Barry, NIAID, unpublished results).
Compound 109 was mostly used in the form of dihydrochloride at five
different doses, and 37--solely as hydrochloride salt at two
doses.
Mice were given a one-time dose of the compounds at concentrations
100, 300 or 1000 mg/kg using the gavage method. Each dose of each
compound consisted of one group of 3 mice. Monitoring of the mice
was done twice a day for the duration of the experiment. Mice
surviving one week post-drug administration were sacrificed;
critical organs were aseptically removed and observed for
abnormalities and evidence of drug toxicity. The MTD (mg/kg) is the
highest dose that results in no lethality/tissue abnormality.
Methods:
1. Treatment of mice: C57BL/6 female mice (6-8 weeks in age) are
given a one-time dose of the compound at concentrations 100, 300 or
1000 mg/kg using the gavage method. The compounds are dissolved in
the appropriate concentration of ethanol in distilled water and
administered in a volume of 0.2 ml per mouse. 2. Observation of
mice: Mice will be observed 4 and 6 hours post administration, then
twice daily for one week. Survival and body weight of mice will be
closely monitored throughout the study. 3. Assessment of drug
toxicity: Mice exhibiting signs of any abnormal appearance or
behavior or those remaining in a group in which other mice did not
survive to day 7 will be sacrificed for assessment of drug
toxicity. Critical organs will be aseptically removed and observed;
tissues from the liver, heart, and kidneys are extracted and placed
into 10% formalin solution. These fixed tissues are sectioned and
examined for abnormalities resulting from drug toxicity.
These studies indicate that the maximum tolerated dose for the
compound 109 is 600 mg/kg (Table 16). No visible changes in organs
were observed. Dose 800 mg/kg was fatal: out of a group of 3 mice,
two animals died within 3 days (Table 17). Compound 37 was well
tolerated at doses 100 and 300 mg/kg. No visible changes in organs
were observed. Additional experiments to evaluate maximum tolerated
dose and in vivo efficacy for the compound 37 are being
conducted.
TABLE-US-00016 TABLE 16 Determination of a maximum tolerated dose
for the compounds 109 and 37 in mice. 109 at 109 at 109 at 109 at
37 at 100 mg/kg 300 mg/kg 600 mg/kg 1000 mg/kg 100 mg/kg Day of Day
of Day of Day of Day of Day Mice death Mice death Mice death Mice
death Mice death Apr. 08, 2003 1 3 3 3 3 2-4 h 1 Apr. 09, 2003 2 3
3 3 2 2 2 Apr. 10, 2003 3 3 3 3 2 2 Apr. 11, 2003 4 3 3 3 1 4 2
Apr. 13, 2003 6 3 3 3 0 6 2 Apr. 14, 2003 7 3 3 3 -- 2
TABLE-US-00017 TABLE 17 Determination of a maximum tolerated dose
for the compounds 109 and 37 in mice. 109 as HCl 109 as TFA 37 at
37 at salt at salt at 100 mg/kg 300 mg/kg 800 mg/kg 800 mg/kg Day
of Day of Day of Day of Date Day Mice death Mice death Mice death
Mice death Apr. 29, 2003 1 3 3 3 1 Apr. 30, 2003 2 3 3 2/1 2 1 May
01, 2003 3 3 3 1/1 3 1 May 02, 2003 4 3 3 1 1 05.03. 5 3 3 1 1
05.04 6 3 3 1 1 May 05, 2003 7 3 3 1 1
EXAMPLE XIV
Pharmacokinetic Studies of the Compounds 37, 59, and 109
Initially, analytical methods for determination of the compounds
had been developed that allowed to carry out all the PK
experiments, see FIG. 29. Here is a brief description of the
experiment: (1) plasma spiked with tested compounds and 10 uL of
Terfenadine or plasma samples (200 uL) added; (2) ACN (2 mL) added
to precipitate protein and spin at 2,500 rpm; (3) evaporate
supernatant to dryness; (4) add 200 uL of the diluting solvent:
methanol (with 0.1% of trifluoroacetic acid): ammonium acetate
(80/20); (5) vortex, spin, and use supernatant; (6) run LC/MS/MS on
Sciex API 3000.
Biostability studies of the compounds in plasma were carried out
using concentrations 1 and 15 mg/ml. The compounds were incubated
for 1, 2, 3 & 6 hr at 37.degree. C. (Table 18). In addition, it
was found that all tested compounds were stable in plasma at
24.degree. C., pH 2 and 7.4 up to 24 hr.
TABLE-US-00018 TABLE 18 Biostability of tested compounds in plasma.
Comp. Human Dog Rat Mouse 37 20%.dwnarw. stable 35% .dwnarw. stable
59 stable stable stable stable 109 30% .dwnarw. 40% .dwnarw. stable
stable
Pilot PK study of the compounds 37, 59, and 109 in mice was
conducted using a cassette dosing: all the three analogs were
formulated together in saline at 1.5 mg/mL, and administered to
mice simultaneously orally at 25 mg/kg, peritoneally at 6 mg/kg,
and intravenously. It was found that doses 15 and 7.5 mg/kg caused
death of mice, 3.75 mg/kg appeared lethargic immediately after
dosing but then appeared normal appearance a few minutes later; 3
mg/kg displayed no adverse reactions and hence was used as
intravenous dose. Obtained data are presented on FIGS. 30, 31, and
32 (tested compounds were studied under the NCI' indexation NSC)
and summarized in Table 19.
TABLE-US-00019 TABLE 19 PK Parameters of tested compounds 37, 59,
and 109 after a cassette dosing to mice. N/A - not detectable.
Route i.v. i.v. i.v. i.p. i.p. i.p. p.o. p.o. p.o. Compounds 37 59
109 37 59 109 37 59 109 Dose (mg/kg) 3 3 3 6 6 6 25 25 25 AUC (ng
h/mL) 954 384 1006 1372 272 1099 1602 169 655 Cmax (ng/mL) 970 296
1192 630 217 935 263 28.7 227 T1/2 (h) 4.8 6.4 5.5 4.9 9.7 4.4 N/A
N/A N/A CL (mL/kg/h) 3530 8043 3240 Bioavailability (%) 72 35 55
3.3 0.9 2.7 Urine excretion (%) .71 1.9 .92 <0.01 <0.01
<0.01 N/A N/A N/A
Conducted pharmacokinetic studies indicated that compound 59 (NSC
722040 by the NCI index) has relatively poor PK profiling (AUC,
Cmax) and further testing of this compound was abandoned. Based on
preliminary toxicity data compound 37 was also ruled out as
possible candidate. Therefore, compound 109 (NSC 722041 by the NCI)
was selected for further PK analyses.
It has been shown that compound SQ109 reaches and exceeds its
Minimum Bactericidal Concentration MBC (313 ng/ml) in plasma when
administered either iv or inatraveneously orally (p.o.), has a
half-life of 5.2 h, and has total clearance less than hepatic blood
flow (FIG. 33, Table 20).
TABLE-US-00020 TABLE 20 Pharmacokinetic parameters of the compound
109. Parameters i.v. p.o. Dose (mg/kg) 3 25 AUC (ng h/mL) 792 254
T.sub.1/2 el (h) 3.5 5.2 C.sub.max (ng/mL) 1038 135 T.sub.max (h) 0
0.31 CL (mL/kg/h) 3788 Vd.sub.ss (mL/kg) 11826 Bioavailability
3.8
Its oral bioavailability is only 3.8% when administered p.o but
this is explained by its unique tissue distribution pattern. Tissue
distribution studies have demonstrated that SQ109 primarily
distributes into the lungs and spleen (FIGS. 34 and 35), which is
highly advantageous for a infection that characteristically
manifests as a lung disease.
By using an ultracentrifugation method, it was found that plasma
protein binding of the compound 109 is concentration dependent and
varies from 15% (20 ng/ml) to 74% (200 ng/ml) to 48% (2000 ng/ml).
After i.v. dosing (3 mg/kg) the compound distributes between plasma
and red blood cells in a ratio 70.6:29.4.
Little is known of the fate of the compound in the body, since the
total amount of the compound after excretion (urine and feces) does
not exceed 3% of the delivered dose (Table 2).
TABLE-US-00021 TABLE 21 Amounts of the compound 109 cumulatively
excreted in mouse urine and feces following single administration.
Period after dosing (h) Dose/ Total Route Samples 0-4 4-8 8-24
24-32 0-32 3 mg/kg Urine <0.01 <0.01 0.03 0.01 0.04 i.v.
Feces <0.01 0.01 0.04 <0.01 0.06 25 mg/kg Urine -- -- -- --
p.o. Feces 0.48 0.31 1.12 0.08 2.0
Initial attempts to identify metabolites of the compound 109 in
urine, did not provide evidence of breakdown products, FIG. 36. For
example, there was no evidence for the formation of conjugated
metabolites (M+521) in the mouse urine during first 24 hr following
compound's administration, FIG. 37. Conjugated metabolites are
products of the typical metabolic pathway N-glucoronidation formed
by reaction with glucuronic acid (D. A. Williams and T. L. Lemke in
Foye's Principals of Medicinal Chemistry, 5.sup.th Ed., p.
202).
EXAMPLE XV
In Vitro Pharmacokinetic Studies of Compound 109
In vitro Pharmacology and early ADMET (Absorption, Distribution,
Metabolism, Excretion, Toxicity) studies of the compound 109 were
contracted out to CEREP (15318 NE 95.sup.th Street, Redmond, Wash.
98052, USA, www.cerep.com, tel 425 895 8666) under a Service
Agreement and included testing against 30 standard receptors (see
CEREP Tables 22 and 23, provided in FIGS. 38 and 39, five CYP450
enzymes, HERG (K+ channel), aqueous solubility, predicted
intestinal permeability, and metabolic stability (data presented in
FIG. 40 Tables 24(a-m)).
EXAMPLE XVI
Bis(2-Adamantyl)ethylenediamine, SQBisAd
##STR00079##
Compounds with the best Selectivity Indexes, such as 109, 58, 73,
78, (Table 15) and good in vivo data share the same adamantane
fragment (FIG. 20). A compound that would have solely this fragment
(on both sides of the ethylene linker) was contemplated. During
preparation of targeted 100,000 compound library of ethambutol
analogues, 70,000 compounds were proven to be formed, but 30,000
were failures. This particular compound was not initially detected
perhaps because it was synthesized in very low yield or because it
was never made due to steric factors.
In the synthetic scheme used for preparation of the library Scheme
1 (FIG. 41), sterically hindered amines on the second step rarely
gave products. Analyzing MS data for a number of original plates it
can be stated that 2-adamantanamine when used as R.sub.1NH.sub.2
seldom yield desirable products and this can be explained because
of existence of sterically hindered reaction site on the step 2 or
step 3 of the synthesis Scheme 2 (FIG. 41).
Compound SQBisAd can be prepared by "wet chemistry" using the same
route, Scheme 3 (FIG. 41), it is documented that 2-adamantamine
(used as commercially available hydrochloride) does provide
products when used on the 1 and 2 steps. Due to the symmetrical
nature, this compound can be synthesized by alternative routes. We
have prepared SQBisAd by reductive alkylation of ethylnediamine by
2-adamantanone using sodium cyanoborohydride. Final product
(without additional purification) demonstrated MIC (Minimal
Ihibitory Concentration) equal or better than compound 109.
EXAMPLE VIII
Generating the Diamine Library with a Modified Linker
General Methods: All reagents were purchased from Sigma-Aldrich.
Rink acid resin was purchased from NovaBiochem, Inc. Solvents
acetonitrile, dichloromethane, dimethylformamide, ethylene
dichloride, methanol, and tetrahydrofuran were purchased from
Aldrich and used as received. Solid phase syntheses were performed
on Quest 210 Synthesizer (Argonaut Technologies) and combinatorial
chemistry equipment (Whatman Polyfiltronics and Robbins
Scientific). Evaporation of the solvents was done using SpeedVac
AES (Savant). Mass spectra data were obtained by Electrospray
Ionization technique on Perkin Elmer/Sciex, API-300, TQMS with an
autosampler.
The activation of the Rink-resin, the addition of the amine, and
the acylation step were carried out in 10 ml tubes using the Quest
210 Synthesizer. Removal of the FMOC group, reductive alkylation
reaction with carbonyl compounds, the reduction with Red-A1, and
the cleavage from the solid support were carried out in 96-deep (2
ml) well, chemically resistant plates.
Step 1. Activation of the Rink-Acid Resin.
A suspension of the Rink-acid resin (coverage of 0.43-0.63 mmol/g),
6 g (up to 3.78 mmol), in 80 ml of 2:1 mixture of dichloromethane
and THF was disitrubuted into 20 tubes, 4 ml per tube, filtered and
washed twice with THF. A solution of triphenylphosphine (5.7 g,
21.75 mmol) in 40 ml of THF was added, 2 ml/tube, followed by the
addition of a solution of hexachloroethane (5.09 g, 21.45 mmol) in
20 ml of ThF, 1 ml/tube. After 6 h the resins were washed with TIF
(2.times.4 ml) and dichloromethane (2.times.4 ml).
Step 2. Addition of the First Amine.
Each tube was charged with 3 ml of dichloroethane, EtNiPr.sub.2,
(0.2 ml, 1.15 mmol), and the corresponding amine (1 mmol). (When a
selected amine was a solid, it was added as a solution or a
suspension in DMF). Dichloroethane was added to each tube to fill
up the volume 4 ml. The reaction was carried for 8 h at 45.degree.
C. and 6-8 h at room temperature. The resins were filtered, washed
with a 2:1 mixture of dichloromethane and methanol (1.times.4 ml),
then with methanol (2.times.4 ml), and suck dry.
Step 3. Acylation with Fmoc Protected Amino Acid.
The resins were pre-washed with dichloromethane (2.times.4 ml).
Each tube was charged with 2 ml of dichloromethane, HATU (2 mol
excess to loaded resin, 0.14 g, 0.39 mmol, dissolved in 1 ml of
DMF), and 0.47 mmol (2.5 mol excess to loaded resin) of amino acid
dissolved in 1 ml of DMP, and allowed to stir for 8 h at 45.degree.
C. and 6-8 h at room temperature. After 16 h the resins were
filtered, washed with 1:1 mixture of DMF and dichloromethane
(1.times.3 ml), dichloromethane (1.times.3 ml) and acylation was
repeated with the same amount of reagents. At the end, the resins
were filtered, washed with 1:1 mixture of DMF and dichloromethane
(1.times.3 ml), and methanol (3.times.3 ml), sucked dry (on Quest)
for 30 min and transferred into vials (one resin per vial), and
dried in a desiccator under vacuum for 1 h. After this step all
resins were subjected for quality control using MS spectra.
Step 4. Alkylation of the Amino Group.
Deprotection. Ten prepared resins from the first three steps were
pooled together, leaving approximately 0.05 g of each in the
individual vials for all necessary deconvolutions. A suspension of
the resin mixture (2.0-2.5 g) in 100 ml of a 2:1 mixture of
dichloromethane and THF was distributed into two 96-well
filterplates and filtered using a filtration manifold. The reaction
plates were transferred into combiclamps, and 0.2 ml of 20%
solution of piperidine in DMF was added to remove Fmoc protecting
group and allowed to stay for 10 min. After 10 min plate was
filtered, washed with 0.2 ml of DMF, and deprotection was repeated
with 0.2 ml of 20% solution of piperidine in DMF and allowed to
stay for 20 min. After that plate was filtered, washed with DMF
(0.2 ml per well) and dichloromethane (2.times.0.5 ml per
well).
Reaction with the carbonyl compounds. Each well in row A on the
reaction plate was charged with 0.1 ml of dichloromethane, 0.08 ml
of .about.1.0M solution of appropriate acid in DMF from master
plate, 0.05 ml DMF solution of PyBrop, (0.015 g, 0.03 mmol, 2.5 mol
excess to loaded resin) and 0.05 ml of EtNir.sub.2 in
dichloromethane (0.022 ml, 0.13 mmol, 10 mol excess to loaded
resin). Each well in rows B through H was charged with 0.1 ml of
THF, 0.160 ml of 1.0 M solution of appropriate aldehyde or ketone
in DMF from master plate and allowed to react for 30 min. After 30
min 0.075 ml (0.075 mmol) of 1.0 M solution of NaBCNH.sub.3 were
added. The reaction plates were sealed and kept at RT for 72 h. At
the end, the resins were filtered, washed with THF, DCM (1.times.1
ml), methanol (2.times.1 ml) and dried in desiccator under vacuum
for 2 h.
Step 5. Reduction with Red-A1.
The reaction plates were placed into combiclamps. A 1:6 mixture of
Red-A1 (65+w % in toluene) and TMF was added, 0.6 ml per well (0.28
mmol of Red-A1 per well), and allowed to react for 4 h. After the
reaction completion the resins were filtered, washed with THF
(2.times.1 ml), methanol (3.times.1 ml) and dried in the filtration
manifold.
Step 6. Cleavage.
This step was carried out using a cleavage manifold. The reaction
plates (placed on the top of the collection plates in this
manifold) were charged with a 10:85:5 mixture of TFA,
dichloromethane, and methanol, 0.5 ml per well. After 15 min, the
solutions were filtered and collected into proper wells of the
collection plates. The procedure was repeated. Solvents were
evaporated on a speedvac, and the residual samples were ready for
testing.
Deconvolution Example.
Deconvolution of the active wells was performed by re-synthesis of
discrete compounds, from the archived FMOC-protected
O-aminoacetamide resins (10 resins, 0.05-0.10 g each), which were
set aside at the end of the acylation step before the pooling. Each
resin was assigned a discrete column (1, or 2, or 3, etc.) in a
96-well filterplate, and was divided between X rows (A, B, C, etc),
where X is the number of hits discovered in the original screening
plate. To each well, in a row, a selected carbonyl compound
(present in the hit) was added along with other required reagents:
the first selected carbonyl compound was added to the resins in the
row "A", the second carbonyl compound--to the resins in the row
"B", the third carbonyl compound--to the resins in the row "C",
etc. A lay-out of a representative 96-well deconvolution plate is
shown in Table 28, FIG. 52.
The reaction plates were sealed and kept at RT for 72 h. At the
end, the resins were filtered, washed with THF, DCM (1.times.1 ml),
methanol (2.times.1 ml) and dried in desiccator under vacuum for 2
h. Reduction and cleavage were performed according to steps 5 and 6
of the synthetic protocol. The product wells from the cleavage were
analyzed by ESI-MS (Electrospray Ionization Mass Spectroscopy) to
ensure the identity of the actives, and were tested in the MIC
assay. A summary of the ESI-MS data is provided below. A list of
compound hits and structures is provided in Table 30, FIG. 53.
Compound 673
N.sup.2-[(2-methoxy-1-naphthyl)methyl]-3-phenyl-N.sup.1-(3-phenylpropyl)-
propane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 439.2
Compound 674
N.sup.2-[2-(benzyloxy)ethyl]-N.sup.1-(3,3-diphenylpropyl)-4-(methylthio)b-
utane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 463.4.
Compound 675
N.sup.1-(3,3-diphenylpropyl)-4-(methylthio)-N.sup.2-(3-phenylpropyl)butan-
e-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 447.2 Compound
676
N.sup.2-(cyclohexylmethyl)-N.sup.1-(3,3-diphenylpropyl)-4-(methylthio)but-
ane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 425.1 Compound
677
N.sup.1-(3,3-diphenylpropyl)-N.sup.2-(2-ethoxybenzyl)-4-(methylthio)butan-
e-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 463.1 Compound
678
N.sup.1-[2-(benzyloxy)ethyl]-N.sup.1-[(6,6-dimethylbicylo[3.1.1]hept-2-yl-
)methyl]-4-(methylthio)butane-1,2-diamine. Mass spectrum (ESI) m/z
(MH).sup.+ 405.3 Compound 679
N.sup.1-[(6,6-dimethylbicyclo[3.1.1]hept-2-yl)methyl]-4-(methylthio)-N-2--
(3-phenylpropyl)butane-1,2-diamine. Mass spectrum (ESI) m/z
(MH).sup.+ 389.5 Compound 680
N.sup.2-(2-chloro-4-fluorobenzyl)-4-methyl-N.sup.1-(4-methylbenzyl)pentan-
e-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 363.3, 365.5;
(MCH.sub.3CN) 403.3, 405.3. Compound 681.
N.sup.2-[2-(benzyloxy)ethyl]-N.sup.1-[2-(4-methoxyphenyl)ethyl]-4-methylp-
entane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 385.1.
Compound 682.
N.sup.1-[3-(4-chlorophenoxy)benzyl]-N.sup.1-[2-(4-methoxyphenyl)ethy-
l]-4-methylpentane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+
467.1, 469.2. Compound 683.
N.sup.2-(4-isopropylbenzyl)-N.sup.1-[2-(4-methoxyphenyl)ethyl]-4-methylpe-
ntane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 383.3
Compound 684.
N.sup.1-[2-(4-methoxyphenyl)ethyl]-4-methyl-N.sup.2-[(2E)-3-phenylprop-2--
enyl]pentane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 367.3;
[M-(CH.sub.2CH.dbd.CHPh).sub.2H]+251. Compound 685
N.sup.2-[2-(benzyloxy)ethyl]-4-methyl-N.sup.1-(3-phenylpropyl)pentane-1,2-
-diamine. Mass spectrum (ESI) m/z(MH).sup.+ 369.1. Compound 686.
AP-(2-chloro-4-fluorobenzyl).sub.4-methyl-N.sup.1-(3-phenylpropyl)pentane-
-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 377.2, 378.9.
Compound 687.
N.sup.2-[3-(4-chlorophenoxy)benzyl]-4-methyl-N'-(3-phenylpropyl)pent-
ane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 451.1, 453.3.
Compound 688.
N.sup.2-(4-isopropylbenzyl).sub.4-methyl-N.sup.1-(3-phenylpropyl)pen-
tane-1,2-iamine. Mass spectrum (ESI) m/z (MH).sup.+ 367.3. Compound
689
4-methyl-N.sup.2-[(2E-3-phenylprop-2-enyl]-N-(3-phenylpropyl)pentane-1,2--
diamine. Mass spectrum (ESI) m/z (MH).sup.+ 351.2. Compound 690
N.sup.2-(2-ethoxybenzyl).sub.4-methyl-N'-(3-phenylpropyl)pentane-1,2-diam-
ine. Mass spectrum (ESI) m/z (MH).sup.+ 369.1. Compound 691.
NW-decahydronaphthalen-2-yl-Nd-[2-(4-fluorophenyl)ethyl]-3-thien-3-ylprop-
ane-1,2-diamine. Mass spectrum (ESI) m/z (MH).sup.+ 415.3.
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